Synthetic membrane anchors

ABSTRACT

The invention relates to synthetic molecules that spontaneously and stably incorporate into lipid by-layers, including cell membranes. Particularly, although not exclusively, the invention relates to the use of these molecules as synthetic membrane anchors or synthetic molecule constructs to effect qualitative and quantitative changes in the expression of cell surface antigens.

FIELD OF INVENTION

The invention relates to synthetic molecules that spontaneously andstably incorporate into lipid bi-layers, including cell membranes.Particularly, although not exclusively, the invention relates to the useof these molecules as synthetic membrane anchors or synthetic moleculeconstructs to effect qualitative and quantitative changes in theexpression of cell surface antigens.

BACKGROUND

Cell surface antigens mediate a range of interactions between cells andtheir environment. These interactions include cell-cell interactions,cell-surface interactions and cell-solute interactions. Cell surfaceantigens also mediate intra-cellular signalling.

Cells are characterised by qualitative and quantitative differences inthe cell surface antigens expressed. Qualitative and quantitativechanges in the cell surface antigens expressed alter both cell function(mode of action) and cell functionality (action served).

Being able to effect qualitative and/or quantitative changes in thesurface antigens expressed by a cell has diagnostic and therapeuticvalue. Transgenic and non-transgenic methods of effecting qualitativeand/or quantitative changes in the surface antigens expressed by a cellare known.

Protein painting is a non-transgenic method for effecting qualitativeand/or quantitative changes in the surface antigens expressed by a cell.The method exploits the ability of GPI linked proteins to spontaneouslyanchor to the cell membrane via their lipid tails. The method describedin the specification accompanying international application no.PCT/US98/15124 (WO 99/05255) includes the step of inserting a GPI linkedprotein isolated from a biological source into a membrane. IsolatedGPI-anchored proteins are stated as having an unusual capacity toreintegrate with a cell-surface membrane.

Cells exist in an aqueous environment. The cell membrane is a lipidbilayer that serves as a semi-permeable barrier between the cytoplasm ofthe cell and this aqueous environment. Localising antigens to the cellsurface may also be achieved by the use of glycolipids as membraneanchors.

The method described in the specification accompanying internationalapplication no. PCT/NZ02/00214 (WO 03/034074) includes the step ofinserting a controlled amount of glycolipid into a membrane. The amountof glycolipid inserted is controlled to provide cells with a desiredlevel of antigen expression.

The method described in the specification accompanying internationalapplication no. PCT/NZ03/00059 (WO 03/087346) includes the step ofinserting a modified glycolipid into a membrane as a “membrane anchor”.The modified glycolipid provides for the localisation of antigens to thesurface of the cell or multicellular structure. New characteristics maythereby be imparted on the cell or multicellular structure.

These methods typically include the isolation of a glycolipid orglycolipid-linked antigen from a biological source. The isolation ofglycolipids or glycolipid-linked antigens from biological sources iscostly, variable and isolatable amounts are often limited. Obtainingreagents from zoological sources for therapeutic use is particularlyproblematic, especially where the reagent or its derivative products areto be administered to a human subject.

Synthetic molecules for which the risk of contamination withzoo-pathogenic agents can be excluded are preferred. Syntheticcounterparts for naturally occurring glycolipids and syntheticneo-glycolipids have been reported. However, for a synthetic glycolipidto be of use as a membrane anchor it must be able to spontaneously andstably incorporate into a lipid bi-layer from an aqueous environment.The utility of synthetic glycolipids in diagnostic or therapeuticapplications is further limited to those synthetic glycolipids that willform a solution in saline.

Organic solvents and/or detergents used to facilitate the solubilizationof glycolipids in saline must be biocompatible. Solvents and detergentsmust often be excluded or quickly removed as they can be damaging tosome cell membranes. The removal of solvents or detergents from suchpreparations can be problematic.

Damage to cell membranes is to be avoided especially where the supply ofcells or multicellular structures is limited, e.g. embryos, or the cellsare particularly sensitive to perturbation, e.g. hepatocytes.

There exists a need for water soluble synthetic molecules that arefunctionally equivalent to naturally occurring glycolipids andglycolipid-linked antigens in respect of their ability to spontaneouslyand stably incorporate into lipid bi-layers, including cell membranes.

Providing such synthetic molecules would obviate the limitations ofglycolipids and glycolipid- linked antigens isolated from biologicalsources and facilitate being able to effect qualitative and/orquantitative changes in the surface antigens expressed by a cell.

It is an object of this invention to provide such synthetic moleculesand a method for their preparation. It is a further object of thisinvention to provide synthetic molecules for use in diagnostic andtherapeutic applications. The preceding objects are to be readdisjunctively with the object to at least provide the public with auseful choice.

STATEMENTS OF INVENTION

In a first aspect the invention consists in a synthetic membrane anchoror synthetic molecule construct of the structure F—S₁-S₂-L where:

-   -   F is selected from the group consisting of carbohydrates;    -   S₁-S₂ is a spacer linking F to L; and    -   L is a lipid selected from the group consisting of diacyl- and        dialkyl-glycerolipids, including glycerophospholipids, and        sphingosine derived diacyl- and dialkyl-lipids, including        ceramide.

Preferably L is a lipid selected from the group consisting of diacyl-and dialkyl-glycerolipids, including glycerophospholipids. Morepreferably L is selected from the group consisting of:diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidylglycerol, and diphosphatidyl glycerol derived from one or more oftrans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoicacid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid,cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoicacid or cis-13-docsenoic acid. More preferably the lipid is derived fromone or more cis-destaurated fatty acids. Most preferably L is selectedfrom the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) andrac-1,2-dioleoylglycerol (DOG).

Preferably L is a glycerophospholipid and the molecule includes thesubstructure:

where n=3 to 5, X is H or C, and * is other than H. Preferably n is 3.

Preferably the molecule is water soluble.

Preferably the molecule spontaneously incorporates into a lipid bi-layerwhen a solution of the molecule is contacted with the lipid bi-layer.More preferably the molecule stably incorporates into the lipid bilayer.

Preferably F, S₁, S₂ and L are covalently linked.

Preferably F is selected from the group consisting of naturallyoccurring or synthetic glycotopes.

S₁-S₂ is selected to provide a water soluble synthetic membrane anchoror synthetic molecule construct.

In a first embodiment F is a naturally occurring or synthetic glycotope.Preferably F is a naturally occurring or synthetic glycotope consistingof three (trisaccharide) or more sugar units. More preferably F is aglycotope selected from the group consisting of lacto-neo-tetraosyl,lactotetraosyl, lacto-nor-hexaosyl, lacto-iso-octaosyl, globoteraosyl,globo-neo-tetraosyl, globopentaosyl, gangliotetraosyl, gangliotriaosyl,gangliopentaosyl, isoglobotriaosyl, isoglobotetraosyl, mucotriaosyl andmucotetraosyl series of oligosaccharides. Most preferably F is selectedfrom the group of glycotopes comprising the terminal sugarsGalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ;NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ;Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc;GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; orGalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc.

When F is a glycotope, L is a glycerophospholipid and S₂ is selectedfrom the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO— (adipate),—CO(CH₂)₅CO— and —CO(CH₂)₅NHCO(CH₂)₅CO—, preferably S₁ is aC₃₋₅-aminoalkyl selected from the group consisting of: 3-aminopropyl,4-aminobutyl, or 5-aminopentyl. More preferably S₁ is 3-aminopropyl.

In a second embodiment F is a molecule that mediates a cell-cell orcell-surface interaction. Preferably F is a carbohydrate with anaffinity for a component expressed on a targeted cell or surface. Morepreferably F has an affinity for a component expressed on epithelialcells or extra-cellular matrices. Yet more preferably F has an affinityfor a component expressed on the epithelial cells or the extra-cellularmatrix of the endometrium. Most preferably the component expressed onthe epithelial cells or the extra-cellular matrix of the endometrium canbe a naturally expressed component or an exogenously incorporatedcomponent.

In a third embodiment F is a molecule that mediates a cell-soluteinteraction. Preferably F is a ligand for a binding molecule where thepresence of the binding molecule is diagnostic for a pathologicalcondition. More preferably F is a ligand for an antibody(immunoglobulin).

In specific embodiments the water soluble synthetic membrane anchor orsynthetic molecule construct has the structure:

designated A_(tri)sp-Ad-DOPE (I); the structure:

designated A_(tri)-spsp₁-Ad-DOPE (II); the structure:

designated A_(tri)-sp-Ad-DSPE (III); the structure

designated B_(tri)-sp-Ad-DOPE (VI); the structure:

designated H_(tri)-sp-Ad-DOPE (VII); the structure:

designated H_(di)-sp-Ad-DOPE (VIII); the structure:

designated Galβ_(i)sp-Ad-DOPE (IX); the structure:

designated Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); orthe structure:

designated Fucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

M is typically H, but may be replaced by another monovalent cation suchas Na⁺, K⁺ or NH₄ ⁺.

In a second aspect the invention consists in a method of preparing asynthetic membrane anchor or synthetic molecule construct of thestructure F—S₁-S₂-L including the steps:

-   -   1. Reacting an activator (A) with a lipid (L) to provide an        activated lipid (A-L);    -   2. Derivatising an antigen (F) to provide a derivatised antigen        (F—S₁); and    -   3. Condensing A-L with F—S₁ to provide the molecule;

where:

-   -   A is an activator selected from the group including:        bis(N-hydroxysuccinimidyl), bis(4-nitrophenyl),        bis(pentafluorophenyl), bis(pentachlorophenyl) esters of        carbodioic acids (C₃ to C₇);    -   L is a lipid selected from the group consisting of diacyl- and        dialkyl-glycerolipids, including glycerophospholipids, and        sphingosine derived diacyl- and dialkyl-lipids, including        ceramide.    -   F is selected from the group consisting of carbohydrates; and    -   S₁-S₇ is a spacer linking F to L where S₁ is selected from the        group including: primary aminoalkyl, secondary aliphatic        aminoalkyl or primary aromatic amine; and S₂ is absent or        selected from the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO—        (adipate), and —CO(CH₂)₅CO—.

Preferably the molecule is water soluble.

Preferably the molecule spontaneously incorporates into a lipid bi-layerwhen a solution of the molecule is contacted with the lipid bi-layer.More preferably the molecule stably incorporates into the lipid bilayer.

Preferably F, S₁, S₂ and L are covalently linked.

Preferably F is selected from the group consisting of naturallyoccurring or synthetic glycotopes.

Preferably L is a lipid selected from the group consisting of diacyl-and dialkyl-glycerolipids, including glycerophospholipids. Morepreferably L is selected from the group consisting of:diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidylglycerol, and diphosphatidyl glycerol derived from one or more oftrans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoicacid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid,cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoicacid or cis-13-docsenoic acid. More preferably the lipid is derived fromone or more cis-destaurated fatty acids. Most preferably L is selectedfrom the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) andrac-1,2-dioleoylglycerol (DOG).

Preferably L is a glycerophospholipid and the molecule includes thesubstructure:

where n=3 to 5, X is H or C, and * is other than H. Preferably n is 3.

Preferably A (R—S₂) and S₁ are selected to provide a water solublesynthetic molecule construct.

In a first embodiment F is a naturally occurring or synthetic glycotope.Preferably F is a naturally occurring or synthetic glycotope consistingof three (trisaccharide) or more sugar units. More preferably F is aglycotope selected from the group consisting of lacto-neo-tetraosyl,lactotetraosyl, lacto-nor-hexaosyl, lacto-iso-octaosyl, globoteraosyl,globo-neo-tetraosyl, globopentaosyl, gangliotetraosyl, gangliotriaosyl,gangliopentaosyl, isoglobotriaosyl, isoglobotetraosyl, mucotriaosyl andmucotetraosyl series of oligosaccharides. Most preferably F is selectedfrom the group of glycotopes comprising the terminal sugarsGalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ;NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ;Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc;GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; orGalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc.

When F is a glycotope, L is a glycerophospholipid and S₂ is selectedfrom the group including: —CO(CH₂)₃CO—, —CO(CH₂)₄CO— (adipate),—CO(CH₂)₅CO— and —CO(CH₂)₅NHCO(CH₂)₅CO—, preferably S₁ is aC₃₋₅-aminoalkyl selected from the group consisting of: 3-aminopropyl,4-aminobutyl, or 5-aminopentyl. More preferably S₁ is 3-aminopropyl.

In a second embodiment F is a molecule that mediates a cell-cell orcell-surface interaction. Preferably F is carbohydrate with an affinityfor a component expressed on a targeted cell or surface. More preferablyF has an affinity for a component expressed on epithelial cells orextra-cellular matrices. Yet more preferably F has an affinity for acomponent expressed on the epithelial cells or the extra-cellular matrixof the endometrium. Most preferably the component expressed on theepithelial cells or the extra-cellular matrix of the endometrium can bea naturally expressed component or an exogenously incorporatedcomponent.

In a third embodiment F is a molecule that mediates a cell-soluteinteraction. Preferably F is a ligand for a binding molecule where thepresence of the binding molecule is diagnostic for a pathologicalcondition. More preferably F is a ligand for an antibody(immunoglobulin).

In specific embodiments the water soluble synthetic molecule constructhas the structure:

designated A_(tri)-sp-Ad-DOPE (I); the structure:

designated A_(tri)-spsp₁-Ad-DOPE (II); the structure:

designated A_(tri)-sp-Ad-DSPE (III); the structure

designated B_(tri)-sp-Ad-DOPE (VI); the structure:

designated H_(tri)sp-Ad-DOPE (VII); the structure:

designated H_(di)sp-Ad-DOPE (VIII); the structure:

designated Galβ_(i)-sp-Ad-DOPE (IX); the structure:

designated Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); orthe structure:

designated Fucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

M is typically H, but may be replaced by another monovalent cation suchas Na⁺, K⁺ or NH₄ ⁺.

In a third aspect the invention consists in a water soluble syntheticmembrane anchor or synthetic molecule construct prepared by a methodaccording to the second aspect of the invention.

In a fourth aspect the invention consists in a method of effectingqualitative and/or quantitative changes in the surface antigensexpressed by a cell or multi-cellular structure including the step:

-   -   1. Contacting a suspension of the cell or multi-cellular        structure with a synthetic membrane anchor or synthetic molecule        construct according to the first aspect or third aspect of the        invention for a time and at a temperature sufficient to effect        the qualitative and/or quantitative change in the surface        antigens expressed by the cell or multi-cellular structure.

Preferably the cell or multi-cellular structure is of human or murineorigin.

Preferably the concentration of the water soluble synthetic membraneanchor or synthetic molecule construct in the suspension is in the range0.1 to 10 mg/mL.

Preferably the temperature is in the range 2 to 37° C. More preferablythe temperature is in the range 2 to 25° C. Most preferably thetemperature is in the range 2 to 4° C.

In a first embodiment the cell is a red blood cell.

In this embodiment preferably F is selected from the group of glycotopescomprising the terminal sugars GalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ;Galβ; Galα1-3(Fucα1-2)Galβ; NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ;Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc;GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; orGalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc. More preferably F is selectedfrom the group of glycotopes consisting of the oligosaccharidesGalNAcα1-3(Fucα1-2)Galβ and Galα1-3(Fucα1-2)Galβ.

Preferably the synthetic molecule construct is selected from the groupincluding: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II);A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE(VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a second embodiment the multi-cellular structure is an embryo.

In this embodiment preferably F is an attachment molecule where theattachment molecule has an affinity for a component expressed on theepithelial cells or the extra-cellular matrix of the endometrium.

The component expressed on the epithelial cells or the extra-cellularmatrix of the endometrium can be a naturally expressed component or anexogenously incorporated component.

Preferably the synthetic membrane anchor or synthetic molecule constructis selected from the group including: A_(tri)-sp-Ad-DOPE (I);A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE(VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII);Galβ_(i)sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a third embodiment the cell is red blood cell.

In this embodiment preferably F is a ligand for a binding molecule wherethe presence of the binding molecule is diagnostic for a pathologicalcondition. More preferably F is a ligand for an antibody(immunoglobulin).

In a fifth aspect the invention consists in a cell or multi-cellularstructure incorporating a water soluble synthetic membrane anchor orsynthetic molecule construct according to the first or third aspect ofthe invention.

Preferably the cell or multi-cellular structure is of human or murineorigin.

In a first embodiment the cell is a red blood cell incorporating a watersoluble synthetic membrane anchor or synthetic molecule constructselected from the group including: A_(tri)-sp-Ad-DOPE (I);A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE(VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE (VIII);Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a second embodiment the multi-cellular structure is an embryoincorporating a water soluble synthetic membrane anchor or syntheticmolecule construct selected from the group consisting of:A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE(III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII);H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In a sixth aspect the invention consists in a kit comprising a driedpreparation or solution of a water soluble synthetic membrane anchor orsynthetic molecule construct according to the first or third aspect ofthe invention.

Preferably the synthetic membrane anchor or water soluble syntheticmolecule construct according to the first or third aspect of theinvention is selected from the group consisting of: A_(tri)-sp-Ad-DOPE(I); A_(tri)-spsp₁-Ad-DOPE (II); A_(tri)-sp-Ad-DSPE (III);B_(tri)sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE (VII); H_(di)-sp-Ad-DOPE(VIII); Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

In an seventh aspect the invention consists in a kit comprising asuspension in a suspending solution of cells or multi-cellularstructures according to the fifth aspect of the invention.

Preferably the suspending solution is substantially free of lipid.

Preferably the cell or multi-cellular structure is of human or murineorigin.

Preferably the cells are red blood cells that do not naturally expressA- or B-antigen and incorporate a water soluble synthetic membraneanchor or synthetic molecule construct selected from the groupconsisting of: A_(tri)-sp-Ad-DOPE (I); A_(tri)-spsp₁-Ad-DOPE (II);A_(tri)-sp-Ad-DSPE (III); B_(tri)-sp-Ad-DOPE (VI); H_(tri)-sp-Ad-DOPE(VII); H_(di)-sp-Ad-DOPE (VIII); Galβ_(i)-sp-Ad-DOPE (IX);Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII); andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII). More preferably thecells are sensitivity controls.

In a eighth aspect the invention consists in a pharmaceuticalpreparation comprising a dried preparation or solution of a watersoluble synthetic membrane anchor or synthetic molecule constructaccording to the first or fourth aspect of the invention.

Preferably the pharmaceutical preparation is in a form foradministration by inhalation.

Preferably the pharmaceutical preparation is in a form foradministration by injection.

In an ninth aspect the invention consists in a pharmaceuticalpreparation comprising cells or multi-cellular structures according tothe fifth aspect of the invention.

Preferably the cells or multi-cellular structures are of human or murineorigin.

Preferably the pharmaceutical preparation is in a form foradministration by inhalation.

Preferably the pharmaceutical preparation is in a form foradministration by injection.

DETAILED DESCRIPTION

The synthetic molecule constructs of the invention spontaneously andstably incorporate into a lipid bi-layer, such as a membrane, when asolution of the molecule is contacted with the lipid bi-layer. Whilstnot wishing to be bound by theory it is believed that the insertion intothe membrane of the lipid tails of the lipid (L) is thermodynamicallyfavoured. Subsequent disassociation of the synthetic molecule constructfrom the lipid membrane is believed to be thermodynamically unfavoured.Surprisingly, the synthetic molecule constructs identified herein havealso been found to be water soluble.

The synthetic molecule constructs of the invention are used to transformcells resulting in qualitative and/or quantitative changes in thesurface antigens expressed. It will be recognised that thetransformation of cells in accordance with the invention isdistinguished from transformation of cells by genetic engineering. Theinvention provides for phenotypic transformation of cells withoutgenetic transformation.

In the context of this description the term “transformation” inreference to cells is used to refer to the insertion or incorporationinto the cell membrane of exogenously prepared synthetic moleculeconstructs thereby effecting qualitative and quantitative changes in thecell surface antigens expressed by the cell.

The synthetic molecule constructs of the invention comprise an antigen(F) linked to a lipid portion (or moiety) (L) via a spacer (S₁-S₂). Thesynthetic molecule constructs can be prepared by the condensation of aprimary aminoalkyl, secondary aliphatic aminoalkyl or primary aromaticamine derivative of the antigen with an activated lipid. Methods ofpreparing neoglycoconjugates have been reviewed (Bovin, N. Biochem. Soc.Symp., 69, 143-160).

A desired phenotypic transformation may be achieved using the syntheticmolecule constructs of the invention in a one step method or a two stepmethod. In the one step method the water soluble synthetic moleculeconstruct (F—S₁-S₂-L) comprises the surface antigen as F.

In the two step method the synthetic molecule construct (F—S₁-S₂-L)comprises an antigen (F) that serves as a functional group to which asurface antigen can be linked following insertion of the syntheticmolecule construct into the membrane. The functional group can be agroup such as a lectin, avidin or biotin. When used in the two stepmethod the synthetic molecule construct is acting as a syntheticmembrane anchor.

In accordance with the invention the primary aminoalkyl, secondaryaliphatic aminoalkyl or primary aromatic amine and the activator of thelipid are selected to provide a synthetic molecule construct that iswater soluble and will spontaneously and stably incorporate into a lipidbi-layer when a solution of the synthetic molecule construct iscontacted with the lipid bi-layer.

In the context of this description the phrase “water soluble” means astable, single phase system is formed when the synthetic moleculeconstruct is contacted with water or saline (such as PBS) in the absenceof organic solvents or detergents, and the term “solution” has acorresponding meaning.

In the context of this description the phrase “stably incorporate” meansthat the synthetic molecule constructs incorporate into the lipidbi-layer or membrane with minimal subsequent exchange between the lipidbi-layer or membrane and the external aqueous environment of the lipidbi-layer or membrane.

The selection of the primary aminoalkyl, secondary aliphatic aminoalkylor primary aromatic amine and the activator depends on thephysico-chemical properties of the antigen (F) to be linked to the lipid(L).

It will be understood by those skilled in the art that for anon-specific interaction, such as the interaction between a diacyl- ordialkyl-glycerolipid and a membrane, structural and stereo-isomers ofnaturally occurring lipids can be functionally equivalent. For example,it is contemplated by the inventors that diacylglycerol 2-phosphatecould be substituted for phosphatidate (diacylglycerol 3-phosphate).Furthermore it is contemplated by the inventors that the absoluteconfiguration of phosphatidate can be either R or S.

The inventors have determined that to prepare synthetic moleculeconstructs of the invention where the antigen (F) is an oligosaccharideselected from the group of glycotopes for A-, B- and H-antigens of theABO blood groups, the primary aminoalkyl, secondary aliphatic aminoalkylor primary aromatic amine, and the activator should be selected toprovide a spacer (S₁-S₂) with a structure according to one of thosepresented here:

Alternative structures of S₁-S₂ for a water soluble synthetic moleculeconstruct (F-S₁-S₂-L) where F is a carbohydrate (or other antigen) withsimilar physico-chemical properties to the carbohydrate portion of theA-, B- or H-antigens of the ABO blood groups and L is aglycerophospholipid (n, m independently = 2 to 5) S₁ is selected from:S₂ is selected from: —O(CH₂)nNH— —CO(CH₂)_(n)CO— or—CO(CH₂)_(m)NHCO(CH₂)_(n)CO—

It will be understood by one skilled in the art that once the structureof the spacer (S₁-S₂) has been determined for a given class of antigens,the same structure of the spacer can be adopted to prepare syntheticmolecule constructs of other classes of antigen with similarphysico-chemical properties.

For example, the structure of the spacer for synthetic moleculeconstructs (F—S₁-S₂-L) where F is a glycotope of the A-, B- andH-antigens of the ABO blood groups, may be the structure of the spacerselected to prepare synthetic molecule constructs of other antigens withphysico-chemical properties similar to the glycotopes of the A-, B- andH-antigens of the ABO blood groups.

In principle the glycotope of a broad range of blood group relatedglycolipids or glycoproteins could be the antigen (F) of the syntheticmolecule construct F—S₁-S₂-L where S₁-S₂-L is identical or equivalent tothe corresponding portion of the synthetic molecule constructsdesignated A_(tri)-sp-Ad-DOPE (I), A_(tri)-spsp₁-Ad-DOPE (II),A_(tri)-sp-Ad-DSPE (III), B_(tri)-sp-Ad-DOPE (VI), H_(tri)-sp-Ad-DOPE(VII), H_(di)-sp-Ad-DOPE (VIII), Galβ-sp-Ad-DOPE (IX),Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII), andFucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).

The structures of known blood group-related glycolipids andglycoproteins (see references) are provided in the following list:

It will be understood by those skilled in the art that the syntheticmolecule constructs (F—S₁-S₂-L) of the invention where F is anoligosaccharide may be used as “synthetic glycolipids” and substitutedfor glycolipids obtained from biological (botanical or zoological)sources.

In the context of this description of the invention the term“glycolipid” means a lipid containing carbohydrate of amphipathiccharacter including: glycosylated glycerolipids, such as glycosylatedphosphoglycerides and glycosylglycerides; glycosylated sphingolipids(neutral glycolipids) such as glycosylceramides or cerebrosides; andgangliosides (acidic glycolipids).

In the context of this description of the invention the phrase“glycolipid-linked antigen” means a lipid containing carbohydrate inwhich an antigen (e.g. a protein) is linked to the glycolipid via thecarbohydrate portion of the molecule. Examples of glycolipid-linkedantigens include GPI-linked proteins.

It will be understood by those skilled in the art that a glycolipid isitself an antigen. The term and phrase “glycolipid” and“glycolipid-linked antigen” are used to distinguish between naturallyoccurring molecules where the antigen is the glycolipid and naturallyoccurring molecules where the antigen is linked to the glycolipid viathe carbohydrate portion of the glycolipid. By analogy the syntheticmolecule constructs of the invention could be described as both“synthetic glycolipids” and synthetic membrane anchors to the extentthat the antigen may be the synthetic glycolipid per se or attached tothe synthetic glycolipid.

It will be understood by those skilled in the art that the carbohydrateportion of a glycolipid may be modified and linked to other antigens bythe methods described in the specification accompanying theinternational application no. PCT/NZ2003/00059 (published asWO03087346).

In the context of this description of the invention the term “glycotope”is used to refer to the antigenic determinant located on thecarbohydrate portion of a glycolipid. The classification of glycolipidantigens in blood group serology is based on the structure of thecarbohydrate portion of the glycolipid.

In blood group serology it is known that the terminal sugars of theglycotopes of A-antigens are GalNAcα1-3(Fucα1-2)Galβ, and the terminalsugars of the glycotopes of the B-antigens are Galα1-3(Fucα1-2)Galβ.Incorporation into the membrane of RBCs of water soluble syntheticmolecule constructs of the invention where F is GalNAcα1-3(Fucα1-2)Galβor Galα1-3(Fucα1-2)Galβ provides RBCs that are serologically equivalentto A-antigen or B-antigen expressing RBCs, respectively.

The terminal three sugars of the carbohydrate portion of the naturallyoccurring A- or B-antigen are the determinant of the A and B bloodgroupings. The terminal four or five sugars of the carbohydrate portionof the naturally occurring A-antigen are the determinant of the A bloodsub-groupings A type 1, A type 2, etc. Accordingly the RBCsincorporating the synthetic molecule constructs of the invention can beused to characterise and discriminate between blood typing reagents(antibodies) of differing specificity.

Water soluble synthetic molecule constructs of the invention thatexclude a carbohydrate portion are contemplated by the inventors.Antigens other than carbohydrates or oligosaccharides, but with similarphysico-chemical properties, may be substituted for F in the “syntheticglycolipids” described.

Synthetic molecule constructs of the invention that comprise an antigen(F) with differing physico-chemical properties to those of carbohydratesor oligosaccharides are also contemplated by the inventors. Watersoluble synthetic molecule constructs comprising these antigens may beprepared by selecting different spacers.

The advantages provided by the synthetic molecule constructs of thisinvention will accrue when used in the practice of the inventionsdescribed in the specifications for the international application nos.PCT/NO2/00212 (published as WO03/034074) and PCT/NZ03/00059 (publishedas WO03087346). The specifications accompanying these applications areincorporated herein by reference.

The synthetic molecule constructs overcome many of the limitations ofusing natural glycolipids in the practice of these inventions. Aparticular advantage of the synthetic molecule constructs is theirsuperior performance and ability to be used in the transformation ofcells at reduced temperatures, e.g. 4° C.

As described herein not all structures of the spacer (S₁-S₂) willprovide a synthetic molecule construct (F—S₁-S₂-L) that is water solubleand spontaneously and stably incorporate in to a lipid bilayer such as acell membrane. The synthetic molecule constructs designatedA_(tri)sp-lipid (IV) and Atri-PAA-DOPE (V) were determined not to bewater soluble and/or unable to spontaneously and stably incorporate into a lipid bilayer such as a cell membrane.

The invention will now be illustrated by reference to the followingnon-limiting Examples and Figures of the accompanying drawings in which:

FIG. 1 shows Diamed results of Cellstab™ stored cells transformed bynatural A glycolipid transformation solution at (L to R) 10 mg/mL, 5mg/mL, 2 mg/mL, 2 mg/mL* and 1 mg/mL. Antisera used are Albaclone (top)and Bioclone (bottom). (*-transformation solution (containingglycolipids) was not washed out after the incubation, it was left inover night and washed out the next day (day 2).)

FIG. 2 shows Diamed results of Cellstab™ stored cells transformed bynatural B glycolipid transformation solution at (L to R) 10 mg/mL, 5mg/mL, 2 mg/mL, 2 mg/mL* and 1 mg/mL. Antisera used are Albaclone (top)and Bioclone (bottom). (*-transformation solution (containingglycolipids) was not washed out after the incubation, it was left inover night and washed out the next day (day 2)).

FIG. 3 shows FACS analysis following in vitro transformation of humanLe(a-b-) red cells with natural Le^(b)-6 glycolipid over time at threetransformation temperatures, 37° C. (top), 22° C. (middle) and 4° C.(bottom).

FIG. 4 shows Diamed results of cells transformed at 4° C. byA_(tri)sp-Ad-DOPE (I) transformation solution at (L to R): washed 0.08mg/mL; unwashed 0.08 mg/mL; washed 0.05 mg/mL; unwashed 0.05 mg/mL;washed 0.03 mg/mL; and unwashed 0.03 mg/mL. The antisera used wasBioclone anti-A.

FIG. 5 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by A_(tri)sp-Ad-DOPE (I)transformation solution at (L to R): 0.08 mg/mL, 0.05 mg/mL and 0.03mg/mL. The antisera used was Bioclone anti-A.

FIG. 6 shows in the left column Diamed results of cells transformed at4° C. by B_(tri)-sp-Ad-DOPE (VI) transformation solution at (L to R):washed 0.6 mg/mL; unwashed 0.6 mg/mL; washed 0.3 mg/mL; unwashed 0.3mg/mL; washed 0.15 mg/mL; and unwashed 0.15 mg/mL; and in the rightcolumn Diamed results of cells transformed at 4° C. byB_(tri)-so-Ad-DOPE (VI) transformation solution at (L to R): washed 0.08mg/mL; unwashed 0.08 mg/mL; washed 0.05 mg/mL; unwashed 0.05 mg/mL;washed 0.03 mg/mL; and unwashed 0.03 mg/mL. The antisera used wasBioclone anti-B.

FIG. 7 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by B_(tri)-sp-Ad-DOPE (VI)transformation solution at (L to R): 0.6 mg/mL, 0.3 mg/mL and 0.15mg/mL.

FIG. 8 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.07+B 0.3 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.07+B 0.3 mg/mL againstanti-A and anti-B.

FIG. 9 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.07+B 0.3 mg/mL against anti-A and anti-B.

FIG. 10 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.07+B 0.2 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.07+B 0.2 mg/mL againstanti-A and anti-B.

FIG. 11 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.07+B 0.2 mg/mL against anti-A and anti-B.

FIG. 12 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.06+B 0.3 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.06+B 0.3 mg/mL againstanti-A and anti-B.

FIG. 13 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.06+B 0.3 mg/mL against anti-A and anti-B.

FIG. 14 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.06+B 0.2 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.06+B 0.2 mg/mL againstanti-A and anti-B.

FIG. 15 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.06+B 0.2 mg/mL against anti-A and anti-B.

FIG. 16 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.05+B 0.3 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.05+B 0.3 mg/mL againstanti-A and anti-B.

FIG. 17 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.05+B 0.3 mg/mL against anti-A and anti-B.

FIG. 18 shows Diamed results of cells transformed at 4° C. by paralleltransformation with A_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI).Wells 1 and 2 (L to R) contain washed A 0.05+B 0.2 mg/mL against anti-Aand anti-B. Wells 3 and 4 contain unwashed A 0.05+B 0.2 mg/mL againstanti-A and anti-B.

FIG. 19 shows cells that were no longer washed prior to testing. Diamedresults of cells transformed at 4° C. by parallel transformation withA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI). Wells 1 and 2 (L toR) contain unwashed A 0.05+B 0.2 mg/mL against anti-A and anti-B.

COMPARATIVE EXAMPLES

The Comparative Examples do not form part of the invention claimed. TheComparative Examples describe red blood cell transformation with naturalglycolipids.

Comparative Example 1 Preparation of Natural Glycolipids

Purification by HPLC

In the first stage, columns were packed with dry silica (15-25 μm)before each run. Relatively dirty samples could be used in HPLC becausethe silica could be discarded along with the theoretically high level ofirreversibly bound contaminants.

Glycolipids were separated on silica gel with a mobile phase ofincreasing polarity. The program was a linear gradient beginning with100% chloroform-methanol-water 80:20:1 (v/v) and ending with 100%chloroform-methanol-water 40:40:12 (v/v).

The HPLC equipment used was a Shimadzu system capable of pumping andmixing four separate solvents at programmed ratios. As chloroform,methanol and water evaporate at different rates, a program was developedwhereby the solvent components were not mixed prior to entering theHPLC.

The Shimadzu HPLC mixes four different liquids by taking a “shot” fromeach of four bottles in turn. “Shots” of chloroform and water directlynext to each other in the lines may cause miscibility problems. Methanolwas sandwiched in between these two immiscible components. Additionally,the water was pre-mixed with methanol in a 1:1 ratio to further preventproblems with miscibility.

Comparative Example 2 Transformation of Red Blood Cell Transformationwith Natural Glycolipids

Agglutination

Transformation of red blood cells was assessed by agglutination usingthe Diamed-ID Micro Typing System in addition to using conventional tubeserology. Diamed ABO typing cards were not used. The cards used wereNaCl, Enzyme test and cold agglutinin cards, which were not pre-loadedwith any antisera or other reagents. This allowed the use of specificantisera with both methodologies.

TABLE 1 Gel-cards. Manufacturer Catalogue ref Diamed NaCl, Enzyme testand cold agglutinin cards

A comparative trial was carried out between tube serology and the Diamedsystem to establish the performance of the two systems. Cells weretransformed at 25° C. for 4 hours. Seraclone and Alba-clone anti-A serawere used to gauge equivalency. The results are shown in Table 3 below.

TABLE 2 Antisera used in comparison of tube serology with the Diamedsystem. Manufacturer Catalogue ref Lot Expiry Albaclone, SNBTS Anti-A.Z0010770 12 Dec. 2004 Seraclone, Biotest 801320100 1310401 12 Apr. 2003

TABLE 3 Agglutination results comparing tube serology with the Diamedsystem. A glycolipid (mg/mL) 10 5 2 1 0 Tube Albaclone 3+ 2+ 0 0 0Seraclone 3+ 2+ 0 0 0 Diamed Albaclone 2+ 2+ 0 0 0 Seraclone 3+ 2+  1+w+ 0

In this experiment, the Diamed system proved to be more sensitive to theweaker reactions than tube serology with the Seraclone anti-A, but notwith Albaclone. These reagents are formulated differently, and are thusnot expected to perform identically. However, the fact that theSeraclone anti-A tube serology combination did not detect positivity isprobably due to operator interpretation. The weaker reactions arenotoriously difficult to accurately score, and the difference between 1+and 0 can be difficult to discern in tubes.

Optimisation

The variables of glycolipid concentration, incubation temperature,incubation duration, diluent and storage solution were examined fortheir effect on cell health. Efficiency and stability of transformationwas assessed by agglutination with the relevant antibody.

TABLE 4 Tube serology agglutination of natural glycolipid A transformedcells over different times and temperatures. A 10 5 2 1 0.1 0.01 0.0010.0001 0 Seraclone 3+ 2+ 0  0  0 (37° C. for 1.5 hours) Seraclone 4+ 3+2+ 1+ w+ 0 0 0 0 (25° C. for 4 hours)

Glycolipid Concentration

Initial transformation experiments were carried out with a highlypurified (HPLC) Le^(b) glycolipid sample and a less pure blood group Aglycolipid sample. Transformation was performed at 37° C. for 1.5 hours

The A glycolipid sample contained other lipid impurities and thuscomparatively less blood group A molecules by weight than the Le^(b)glycolipid sample of equivalent concentration (w/v). This seems to beborne out by the fact that higher concentrations of the A glycolipidthan the Le^(b) glycolipid were required to produce equivalentagglutination scores (see Table 6).

The level of impurity in the A glycolipid sample may also havecontributed to the lower stability over the 62 day period—theA-transformed cells ‘died’ at the highest concentration (having receivedthe largest dose of impurity).

TABLE 5 Anti-A and anti-Le^(b) used in initial testing of naturalglycolipid transformation. Manufacturer Catalogue ref Batch numberExpiry Anti-A Seraclone, Biotest 801320100 1310401 12 Apr. 2003Anti-Le^(b) CSL 12801

TABLE 6 Stability of RBCs transformed with natural A and Le^(b)glycolipid as assessed by tube serology agglutination over the period of62 days. Glycolipid Le^(b) A (mg/mL) Day 1 Day 25 Day 62 Day 1 Day 25Day 62 10 4+ 2-3+  3+ 2+ ? 5 4+ 2-3+  2+ 2+ w+ 2 3+ 1-2+ 0 1+ 0 1 4+  2+0 1+ 0 0.1 3+ 2+ 0 0 0.01 2+ 2+ 0 0 0.001 2+ 2+ 0 0 0.0001 2+ 0  0 0 00  0  0 0 0  0

The above cells were also rated for haemolysis and these results areshown in Table 7 below.

TABLE 7 Haemolysis as assessed visually. Day 1 - in the supernatant ofthe first wash after transformation; Days 25 and 62 - in the cellpreservative solution before the cells are resuspended after storage.Scoring scale is analogous to the 4+ to 0 agglutination scale: hhhh -severely haemolysed, hhh - very haemolysed, hh - moderately haemolysed,h - mildly haemolysed, w - faintly haemolysed and 0 - no haemolysisseen. Glycolipid Haemolysis concentration Le^(b) A (mg/mL) Day 1 Day 25Day 62 Day 1 Day 25 Day 62 10 h 0 h h h dead 5 hh 0 hhh w 0 hh 2 w 0 hhhw 0 hhhhh 1 w 0 hhh h 0 hhhh 0.1 h hhh 0.01 hh 0.001 h 0.0001 h Controlh 0 h h h

These results show that cell haemolysis can be shown to be associatedwith transformation with high concentrations of glycolipid. It isunclear whether the mechanism underlying this is disruption of theplasma membrane by large amounts of glycolipid being inserted, the rateof that insertion, or is possibly due to the quantity of associatedimpurity. However, the results for Le^(b) at day 62 seem to support thefirst explanation.

The Le^(b) sample was highly purified—before being dissolved, it was apowder of pure white colour, and thus it is unlikely that the haemolysiswas due to the deleterious effect of impurities. It is clear to see thatat 62 days, the amount of haemolysis occurring diminishes in line withthe decrease in the glycolipid concentration.

Incubation Temperature

Experiments were carried out to investigate other possible mechanismsfor the reduction of haemolysis of RBCs during the insertion step.Previous experiments had shown that haemolysis was worse at higherglycolipid concentrations than at lower concentrations, and it isthought that haemolysis may also be related to the rate of glycolipidinsertion. Since temperature is believed to affect the rate ofinsertion, experiments were conducted comparing transformation at 37° C.with transformation at room temperature (RT; 25° C.).

Since the rate was expected to slow down as temperature decreased, theincubation period for the RT experiment was 4 hrs. Haemolysis wasassessed visually and scored following insertion. Serology tests werealso performed on the cells. The results are shown in Table 8.

TABLE 8 The effect of incubation temperature on haemolysis andagglutination during insertion of glycolipids into RBC membranes.Haemolysis was scored visually at each of the three washes. HaemolysisRT 37° C. Glycolipid wash wash wash wash Serology (mg/mL) 1 2 3 1 wash 2wash 3 RT 37° C. 10 w 0 0 hh w 0 2+ 2+ 1 w 0 0 hh h vw 1+ w+

Incubation Duration

Incubation at 37° C. was carried out for 1 and 2 hours and its effect oncell health and transformation assessed by agglutination with therelevant antibody.

TABLE 9 Antisera used in the duration of incubation trial. ManufacturerCatalogue ref Batch number Expiry date Albaclone, SNBTS Anti-A. Z001077012 Dec. 2004 Bioclone, OCD Anti-A, DEV01102 — experimental reagentAlbaclone, SNBTS Anti-B Z0110670 01 Jul. 2005 Bioclone, OCD Anti-B,DEV01103 — experimental reagent

TABLE 10 Effect of incubation time on agglutination of cells transformedwith natural glycolipids. Concentration Albaclone BioClone Glycolipid(mg/mL) 1 hour 2 hours 1 hour 2 hours A 10 4+ 4+ 4+ 4+ 5 4+ 4+ 4+ 2+ 24+ 3+ 3+ 2+ 1 3+ 2+ 2+ 2+ 0.5 2+ 2+ 1+ w+ B 10 3+ 2+ 4+ 1+ 5 3+ 2+ 3+ 2+2 2+ 2+ 2+ 1+ 1 1+ w+ 1+ w+ 0.5 1+ w+ w+ w+

These results indicate that increasing the duration of incubation duringnatural glycolipid insertion does not enhance agglutination. In fact,the agglutination scores are reduced after the two hour incubation. Thismay be due to the destabilisation of the membrane or exchange of theglycolipids back into solution.

Diluent

Experiments were also carried out to determine if changing theglycolipid diluent solution could reduce haemolysis. Working strengthPBS was compared with 2×PBS and 2% Bovine Serum Albumin (BSA) in workingstrength PBS. Cells were incubated at 37° C. for 1.5 hours. The resultsare shown in Table 11.

TABLE 11 Study on the effect on haemolysis of changing the glycolipiddiluent solutions during insertion of glycolipids into RBC membranes.Glycolipid concentration Glycolipid Diluent Solution (mg/mL) PBS 2 × PBS2% BSA in PBS 40 Hhh hhh hhh 30 Hhh hhh hhh 20 Hhh hhh hhh 10 Hhh hhhhhh 0 0 0 0

Stability

Once A and B blood group glycolipids had been HPLC purified to anacceptable level, an experiment to find the appropriate concentrationsfor stability trials was carried out.

TABLE 12 Early stability trial of cells transformed with natural Aglycolipid. A Expt Day 10 5 2 1 0.1 0.01 0.001 0.0001 0 1 7 4+ 3-4+  1+0 0 0 0 0 0 2 43 3+ w+ 0 0 0 0 0 0 0 3 50 1+ 0  0 0 4 60 3+ 1+ 0 5 67 w+vw vw 6 74 2+ 0  0 7 81 2+ 1+ 0

TABLE 13 Antisera used in stability trials (Table 14 and Table 15).Manufacturer Catalogue ref Batch number Expiry date Albaclone, SNBTSAnti-A. Z0010770 12 Dec. 2004 Bioclone, OCD Anti-A, DEV01102 —experimental reagent Albaclone, SNBTS Anti-B Z0110670 01 Jul. 2005Bioclone, OCD Anti-B, DEV01103 — experimental reagent

TABLE 14 Tube serology of O RBCs transformed with A glycolipid in orderto establish appropriate concentrations for stability trials. Aglycolipid (mg/mL) Anti-A Expt 10 5 2 1 0.5 0.1 0.01 0.001 0 Alba 1 3+2+ 1+ 0  0 0 0 0 2 4+ 4+ 3+ 2+ w+ Bio 1 3+ 2+ 1+ 0  0 0 0 0 2 4+ 4+ 3+2+ w+ 1 & 2 Transformation at 25° C. for 4 hours

TABLE 15 Tube serology of O RBCS transformed with B glycolipid in orderto establish appropriate concentrations for stability trials. Bglycolipid (mg/mL) Anti-B Expt 10 5 2 1 0.5 0.1 0.01 0.001 0 Alba 1 2+1+ w+ 0 0 0 0 0 2 1+ 1+ w+ 0 w+ Bio 1 3+ 2+ w+ 0 0 0 0 0 2 1+ 1+ w+ 0 w+1 & 2 Transformation at 25° C. for 4 hours

Two sets of cells were transformed with different concentrations ofnatural A glycolipid. Transformation was performed at 25° C. One set ofcells was tested long term, and one set of cells was tested weekly foragglutination. The agglutination results from tube serology and Diamedare shown in Table 16 below. All cells were stored in Cellstab™ inbottles with flat bases. The cells showed minimal to no haemolysis atany time.

TABLE 16 Agglutination results for cells transformed with differentconcentrations of natural A glycolipid. Results were obtained usingAlbaclone anti-A. A glycolipid (mg/mL) 10 5 2 1 0.1 control Long termtesting Day 1 Tube 4+ 3+  2+  1+ +w 0 Diamed 3+ 3+ +w 0 0 0 Day 17 Tube3+ 2+ 0 0 0 Diamed 3+ 2+  1+ 0 0 Weekly testing Day 1 Tube 3+  2+ 0 0Diamed 3+ 0 0 0 Day 8 Tube 1+ 0 0 0 Diamed 3+ 0 0 0 Day 15 Tube 1+ 0 0 0Diamed 3+  2+ 0 0 Day 22 Tube 3+ 0 0 0 Diamed 3+ 0 0 0 Day 29 Tube *+w*0  *0  *0 Diamed *3+  *0  *0  *0 Day 36 Tube * * * *0 Diamed *3+  *0 *0  *0 Day 43 Tube * * * *0 Diamed * * * *0 *Albaclone, while all othersused Seraclone anti-A.

Storage Solution

Comparison of the two cell storage solutions, Celpresol™ (CSL) andCellstab™ (Diamed) was carried out to test their relative abilities tosupport modified RBCs.

The stability of RBCs transformed with blood group A and B antigensolutions of varying concentrations when stored in two different cellpreservative solutions—Cellstab™ and Alsevers™—was trialed.

A and B antisera from two different sources were used in serologytesting.

All cells were tested using the standard tube serology platform up to 42days, at which time the cell agglutination reactions had become toodifficult to score manually (see Table 17 for A results and Table 18 forB results).

Diamed gel-card testing was carried out to day 56 for the Alseversstored cells, and discontinued at day 63 due to fungal contamination(although still returning positive scores).

The Cellstab™ stored cells continued to be tested up to day 70, and werestill viable at this point (see FIG. 1 for A results and FIG. 2 for Bresults).

The reagents used in the stability trial are shown in Table 13.

TABLE 17 Tube serology results of stability trial of cells transformedwith varying concentrations of A glycolipid and stored in eitherCellstab ™ or Alsevers ™ Albaclone Anti-A Bioclone Anti-A (SNBTS)Transformation (OCD - Solution (mg/mL) Developmental reagent) Day Cellstorage solution 10 5 2 2* 1 10 5 2 2* 1 2 Alsevers 4+ 3+ 2+ 1+ w+ 3+ 3+1+ 1+ 0 Cellstab ™ 4+ 4+ 3+ 1+ 1+ 3+ 3+ 2+ 1+ 0 8 Alsevers 4+ 4+ 2+ 1+1+ 2+ 2+ 1+ 1+ 0 Cellstab ™ 4+ 4+ 3+ 2+ 1+ 3+ 3+ 2+ w+ 0 14 Alsevers 4+3+ 2+ 2+ w+ 2+ 1+ w+ vw 0 Cellstab ™ 4+ 3+ 3+ 2+ w+ 3+ 2+ w+ vw 0 21Alsevers 3+ 2+ 2+ 2+ 1+ 2+ 2+ 2+ 1+ 0 Cellstab ™ 3+ 3+ 2+ + ^(‡) 2+ ^(‡)^(‡) ^(‡) 0 28 Alsevers 2+ 2+ 1+ 1+ 0 2+ 2+ 1+ 1+ 0 Cellstab ™ 2+^(‡)2+^(‡) ^(‡) ^(‡) 0 1+ w+ 0 0 0 36 Alsevers 3+ 2+ 2+ 2+ 1+ 3+ 3+ 2+ 1+ 1+Cellstab ™ 3+^(‡) 2+^(‡) ^(‡) ^(‡) ^(‡) 3+^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 42Alsevers 3+ 3+ 1+ w+ 0 2+ 2+ 2+ 1+ 1+ Cellstab ™ 4+^(‡) 4+^(‡) ^(‡) ^(‡)^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 0 *transformation solution (containingglycolipids) was not washed out after the incubation, it was left inover night and washed out the next day. ^(‡)positive cell button, butcells fall off as negative (score assignment impossible).

TABLE 18 Tube serology results of stability trial of cells transformedwith varying concentrations of B glycolipid and stored in eitherCellstab ™ or Alsevers ™. Albaclone Anti-B (SNBTS) Bioclone Anti-B CellTransformation (OCD - storage Solution (mg/mL) Developmental reagent)Day solution 10 5 2 2* 1 10 5 2 2* 1 2 Alsevers 3+ 3+ 1+ 1+ 1+ 2+ 1+ 1+1+ 0 Cellstab ™ 3+ 3+ 2+ 2+ 1+ 2+ 2+ 2+ 1+ w+ 8 Alsevers 1+ 1+ w+ 0 0 00 0 0 0 Cellstab ™ 2+ 1+ w+ 0 1+ 1+ w+ 0 0 14 Alsevers 2+ 2+ 0 w+ 0 0 1+1+ 2+ 0 Cellstab ™ 1+ w+ 0 0 0 2+ 2+ w+ 1+ 1+ 21 Alsevers ^(‡) ^(‡) ^(‡)^(‡) ^(‡) 1 1 ^(‡) ^(‡) ^(‡) Cellstab ™ ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) + + +^(‡) ^(‡) 28 Alsevers 2+ 1+ w+ 0 0 2+ 1+ 2+ 0 0 Cellstab ™ ^(‡) ^(‡)^(‡) 0 0 ^(‡) 0 ^(‡) ^(‡) 0 36 Alsevers 2+ 2+ 2+ 1+ 1+ 2+ 2+ 2+ 1+ 1+Cellstab ™ ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) 42 Alsevers2+ 2+ 2+ 2+ w+ 2+ 2+ 1+ w+ w+ Cellstab ™ ^(‡) ^(‡) ^(‡) ^(‡) ^(‡) ^(‡)^(‡) ^(‡) ^(‡) ^(‡) *transformation solution (containing glycolipids)was not washed out after the incubation, it was left in over night andwashed out the next day. ^(‡)positive cell button, but cells fall off asnegative (score assignment impossible).

FACS Analysis of Glycolipid Insertion

Transformation of human Le(a-b-) red cells with natural Le^(b)-6glycolipid over time at three transformation temperatures (37° C., 22°C. and 4° C.) was performed (FIG. 3). Natural Le^(b)-6 glycolipid wasdissolved in plasma and used to transform RBCs at a final concentrationof 2 mg/mL and a final suspension of 10%.

Reactivity was determined by FACS analysis using a Gamma anti-Leb. (Theserological detection level is around 10² molecules. The insertion ofnatural glycolipids at 4° C. for 8 hours was not detectable byagglutination with antibodies.) Projection of the rate of insertioncurve from FACS analysis did not indicate that the rate of insertion at4° C. would have reached agglutination detection levels within 24 hours.

Low Incubation Temperature

Transformation of RBCs with natural A or B glycolipid was performed at37° C. for 1 hour and 2° C. for varying intervals. Cells wereagglutinated with Bioclone anti-A or Bioclone anti-B. The results areprovided in Tables 19 and 20.

TABLE 19 Diamed results of comparison of natural A glycolipidtransformation at 37° C. for 1 hour and 2° C. for varying intervals.Time Nat A (mg/mL) Temp (hours) 10 5 2 1 0 37° C. 1  3+  3+ 2-3+  2+ 0 2° C. 1 0 0 0 0 0 4 0 0 0 0 0 8 1-2+ 0 0 0 0 24 2-3+  2+ 1-2+ 0 0 48 3+2-3+ 2-3+ 0 0 72 3-4+  3+  2+ 0 0

TABLE 20 Diamed results of comparison of natural B glycolipidtransformation at 37° C. for 1 hour and 2° C. for varying intervals.Time Nat B (mg/mL) Temp (hours) 10 5 2 1 0 37° C. 1 3+ 2-3+  2+ 0 0  2°C. 1 0  0 0 0 0 4 0  0 0 0 0 8 0  0 0 0 0 24 1+ 0 0 0 0 48 2+ 1-2+ 0 0 072 2+  1+ 0 0 0

The rate of transformation is slow for both natural A glycolipid andnatural B glycolipid as demonstrated by the negative agglutinationscores after 1 hour at 2° C. Considerable insertion at 37° C. for thistime interval has been demonstrated.

Natural A glycolipid insertion at 2° C. required 48 hours to reach thesame level of insertion obtainable by transformation at 37° C. Afterthis time further insertion was not observed. Likewise, natural Bglycolipid insertion at 2° C. was not as rapid as transformation at 37°C. The agglutination scores did not improve upon continued incubationand thus seemed to have reached maximal insertion at this time point forthese concentrations.

EXAMPLES

The Examples describe red blood cell transformation with the syntheticmolecule constructs of the invention. In the context of these examplesthe term “synthetic glycolipids” is used to refer to these constructs.

Example 1 Preparation of Synthetic Glycolipids

Materials and Methods

TLC analysis was performed on silica gel 60 F₂₅₄ plates (Merck), thecompounds were detected by staining with 8% of phosphoric acid in waterfollowed by heating at over 200° C. Column chromatography was carriedout on silica gel 60 (0.2-0.063 mm, Merck) or Sephadex LH-20 (Amersham).¹H NMR spectra were acquired on a Bruker DRX-500 spectrometer. Chemicalshifts are given in ppm (8) relative to CD₃OD.

Synthesis of activated1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and1,2-O-distereoyl-sn-glycero-3-phosphatidylethanolamine(DSPE)(glycerophospholipids)

To a solution of bis(N-hydroxysuccinimidyl) adipate (A) (70 mg, 205μmol) in dry N,N-dimethylformamide (1.5 ml) were added DOPE or DSPE (L)(40 μmol) in chloroform (1.5 ml) followed by triethylamine (7 μl). Themixture was kept for 2 h at room temperature, then neutralized withacetic acid and partially concentrated in vacuo.

Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol, 0.2%acetic acid) of the residue yielded the activated lipid (A-L) (37 mg,95%) as a colorless syrup; TLC (chloroform-methanol-water, 6:3:0.5):R_(f)=0.5 (DOPE-A), R_(f)=0.55 (DSPE-A).

¹H NMR (CDCl₃/CD₃OD, 2:1), δ:

DOPE-A—5.5 (m, 4H, 2×(—CH═CH—), 5.39 (m, 1H, —OCH₂—CHO—CH₂O—), 4.58 (dd,1H, J=3.67, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.34 (dd, 1H, J=6.61, J=11.98,—CCOOHCH—CHO—CH₂O—), 4.26 (m, 2H, PO—CH₂ —CH₂—NH₂), 4.18 (m, 2H, —CH₂—OP), 3.62 (m, 2H, PO—CH₂—CH₂ —NH₂), 3.00 (s, 4H, ONSuc), 2.8 (m, 2H,—CH₂ —CO (Ad), 2.50 (m, 4H, 2×(—CH₂ —CO), 2.42 (m, 2H, —CH₂ —CO (Ad),2.17 (m, 8H, 2×(—CH₂ —CH═CH—CH₂ —), 1.93 (m, 4H, COCH₂CH₂ CH₂ CH₂CO),1.78 (m, 4H, 2×(COCH₂CH₂ —), 1,43, 1.47 (2 bs, 40H, 20 CH₂), 1.04 (m,6H, 2 CH₃).

DSPE-A—5.39 (m, 1 H, —OCH₂—CHO—CH₂O—), 4.53 (dd, 1H, J=3.42, J=11.98,—CCOOHCH—CHO—CH₂O—), 4.33 (dd, 1H, J=6.87, J=11.98, —CCOOHCH—CHO—CH₂O—),4.23 (m, 2H, PO—CH₂ —CH₂—NH₂), 4.15 (m, 2H, —CH₂ —OP), 3.61 (m, 2H,PO—CH₂—CH₂ —NH₂), 3.00 (s, 4H, ONSuc), 2.81 (m, 2H, —CH₂ —CO (Ad), 2.48(m, 4H, 2×(—CH₂ —CO), 2.42 (m, 2H, —CH₂ —CO (Ad), 1.93 (m, 4H, COCH₂CH₂CH₂ CH₂CO), 1.78 (m, 4H, 2×(COCH₂CH₂ ), 1.43, 1.47 (2 bs, 40H, 20 CH₂),1.04 (m, 6H, 2 CH₃).

Condensing Activated DOPE (or DSPE) with Aminopropylglycoside.

To a solution of activated DOPE (or DSPE) (A-L) (33 μmol) inN,N-dimethylformamide (1 ml) 30 μmol of Sug-S₁—NH₂ (F—S₁—NH₂) and 5 μlof triethylamine were added. For example, the Sug may be either theaminopropyl glycoside (F—S₁—NH₂) of either GalNAcα1-3(Fucα1-2)Galβtrisaccharide (A-glycotope) (F) or Galα1-3(Fucα1-2)Galβ trisaccharide(B-glycotope) (F).

The mixture was stirred for 2 h at room temperature. Columnchromatography (Sephadex LH-20 in 1:1 chloroform-methanol followed bysilica gel in ethyl acetate-isopropanol-water, 4:3:1 (v/v/v) of themixture typically yielded 85-90% of the synthetic molecule construct,for example, A_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE (VI).

¹H NMR (CDCl₃/CD₃OD, 1:1), δ:

A_(tri)-sp-Ad-DOPE (I)—5.5 (m, 4H, 2×(—CH═CH—), 5.43-5.37 (m, 2H, H-1(GalNHAc) and —OCH₂—CHO—CH₂O—), 5.32 (d, 1H, H-1, J=3.5 H-1 Fuc), 2.50(m, 4H, 2×(—CH₂ —CO), 2.40 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.20 (m, 8H,2×(—CH₂ —CH═CH—CH₂ —), 2.1 (s, 3H, NHAc), 1.92 (m, 2H, O—CH₂CH₂ CH₂—NH),1.8 (m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ ), 1.43, 1.47 (2 bs, 40H,20 CH₂), 1.40 (d, 3H, J=6.6, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃).

A_(tri)-spsp₁-Ad-DOPE (II)—5.5 (m, 4H, 2×(—CH═CH—), 5.43-5.37 [m, 2H,H-1 (GalNHAc) and —OCH₂—CHO—CH₂O—], 5.32 (d, 1H, H-1, J=3.6 H-1 Fuc),2.50 (m, 4H, 2×(—CH₂ —CO), 2.40-2.32 (m, 6H, COCH₂ CH₂CH₂CH₂ CO andCOCH₂ — (sp₁), 2.18 [m, 8H, 2×(—CH₂ —CH═CH—CH₂ —)], 2.1 (s, 3H, NHAc),1.95(m, 2H, O—CH₂CH₂ CH₂—NH), 1.8 [m, 10H, COCH₂CH₂ CH₂ CH₂CO,2×(COCH₂CH₂ — . . . ), —COCH ₂CH₂ (CH₂)₃NH—], 1.68 (m, 2H, CO(CH₂)₃CH₂CH₂NH—), 1.43, 1.47 (2 bs, 42H, 22 CH₂), 1.37 (d, 3H, J=5.6, CH₃ Fuc),1.05 (m, 6H, 2 CH₃).

A_(tri)-sp-Ad-DSPE (III)—5.42-5.38 (m, 2H, H-1 (GalNHAc) and—OCH₂—CHO—CH₂O—), 5.31 (d, 1H, H-1, J=3.5 H-1 Fuc), 2.48 [m, 4H, 2×(—CH₂—CO)], 2.42 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.18 (s, 3H, NHAc), 1.95 (m,2H, O—CH₂CH₂ CH₂—NH), 1.8 [m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂—)], 1.43, 1.47 (2 bs, 56H, 28 CH₂), 1.38 (d, 3H, J=6.6, CH₃ Fuc), 1.05(m, 6H, 2 CH₃).

B_(tri)-sp-Ad-DOPE (VI)—5.5 (m, 4H, 2×(—CH═CH—), 5.42-5.38 [m, 2H, H-1(Gal) and —OCH₂—CHO—CH₂O—], 5.31 (d, 1H, H-1, J=3.7, H-1 Fuc), 2.48 [m,4H, 2×(—CH₂ —CO)], 2.39 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.18 [m, 8H,2×(—CH₂ —CH═CH—CH₂ —)], 1.93 (m, 2H, O—CH₂CH₂ CH₂—NH), 1.8 [m, 8H,COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ —)], 1.43, 1.47 (2 bs, 40H, 20 CH₂),1.36 (d, 3H, J=6.6, CH₃ Fuc), 1.05 (m, 6H, 2 CH₃).

H_(tri)-sp-Ad-DOPE (VII)—5.5 [m, 4H, 2×(—CH═CH—)], 5.4 (m, 1H,—OCH₂—CHO—CH₂O—), 5.35 (d, 1H, H-1, J=3.2, H-1 Fuc), 4.65, 4.54 (2d,J=7.4, J=8.6, H-1 Gal, H-1 GlcNHAc), 4.46 (dd, 1H J=3.18, J=12,—CCOOHCH—CHO—CH₂O—), 4.38-4.28 (m, 2H, H-5 Fuc, CCOOHCH—CHO—CH₂O—), 2.48[m, 4H, 2×(—CH₂ —CO)], 2.40 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.18 [m, 8H,2×(—CH₂ —CH═CH—CH₂ —)], 2.08 (s, 3H,NHAc), 1.92 (m, 2H, O—CH₂CH₂CH₂—NH), 1.82-1.72 [m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ —)], 1.48,1.45 (2 bs, 40H, 20 CH₂), 1.39 (d, 3H, J=6.5, CH₃ Fuc), 1.05 (m, 6H, 2CH₃).

H_(di)-sp-Ad-DOPE (VIII)—5.49 (m, 4H, 2×(—CH═CH—), 5.37 (m, 1H,—OCH₂—CHO—CH₂O—), 5.24 (d, 1H, H-1, J=2.95, H-1 Fuc), 4.46 (d, J=7.34,H-1 Gal), 2.48 [m, 4H, 2×(—CH₂ —CO)], 2.42-2.35 (m, 4H, COCH₂ CH₂CH₂CH₂CO), 2.17 [m, 8H, 2×(—CH₂ —CH═CH—CH₂ —)], 1.95 (m, 2H, O—CH₂CH₂ CH₂—NH),1.81-1.74 [m, 8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ —)], 1.45, 1.41 (2bs, 40H, 20 CH₂), 1.39 (d, 3H, J=6.5, CH₃ Fuc), 1.03 (m, 6H, 2 CH₃).

Galβ-sp-Ad-DOPE (IX)—5.51 [m, 4H, 2×(—CH═CH—)], 5.4 (m, 1H,—OCH₂—CHO—CH₂O—), 4.61 (dd, 1H J=3.18, J=12, —CCOOHCH—CHO—CH₂O—), 4.41(d, J=7.8, H-1 Gal), 4.37 (dd, 1H, J=6.6, J=12, —CCOOHCH—CHO—CH₂O—),2.50 [m, 4H, 2×(—CH₂ —CO)], 2.40 (m, 4H, COCH₂ CH₂CH₂CH₂ CO), 2.20 [m,8H, 2×(—CH₂ —CH═CH—CH₂ —)], 1.97 (m, 2H, O—CH₂CH₂ CH₂—NH), 1.82-1.72 [m,8H, COCH₂CH₂ CH₂ CH₂CO and 2×(COCH₂CH₂ —)], 1.48, 1.45 (2 bs, 40H, 20CH₂), 1.05 (m, 6H, 2 CH₃).

Example 2 Solubility of Synthetic Glycolipids

For use in the transformation of cells the first criterion thatsynthetic glycolipids must satisfy is that they are soluble in aqueoussolvents, e.g. phosphate buffered saline. A number of techniques,including heat and/or sonication, were employed initially in order tomaximise the solubility of the synthetic glycolipids tested (Table 21).

The synthetic glycolipid must also be able to insert into the membraneand be recognisable to the appropriate antibody for transformation to bedetected by agglutination. Initial tests on the molecules were toestablish solubility and thus eliminate those molecules that wereunsuitable for use in the transformation of cells.

The results of these initial tests are provided in Table 22.

TABLE 21 The range of synthetic glycolipid molecules tested. DOPE LipidTails: B_(tri)-sp-Ad-DOPE (VI) A_(tri)-sp-Ad-DOPE (I) Galβ-sp-Ad-DOPE(IX) H_(di)-sp-Ad-DOPE (VIII) H_(tri)-sp-Ad-DOPE (VII)A_(tri)-spsp₁-Ad-DOPE (II) B_(tri)-PAA-DOPE (V) Different Lipid Tails:A_(tri)-sp-lipid (IV) A_(tri)-sp-Ad-DSPE (III)

TABLE 22 Solubility of synthetic glycolipids in hot PBS andtransformation ability. Detectable transformation Synthetic Watersolubility ability A_(tri)-sp-lipid (IV) No No B_(tri)-PAA-DOPE (V) NoNo B_(tri)-sp-Ad-DOPE (VI) Yes Yes A_(tri)-sp-Ad-DOPE (I) Yes YesGalβ-sp-Ad-DOPE (IX) Yes No H_(di)-sp-Ad-DOPE (VIII) Yes NoH_(tri)-sp-Ad-DOPE (VII) Yes Yes A_(tri)-spsp₁-Ad-DOPE (II) Yes YesA_(tri)-sp-Ad-DSPE (III) Yes Yes

The lack of detectable transformation for Galβ-sp-Ad-DOPE (IX) andH_(di)-sp-Ad-DOPE (VIII) was thought to be due to the inability of theantibody to recognise the glycotope of these synthetic molecules.A_(tri)-sp-lipid (IV) has a single rather than a diacyl tail and it wasproposed that there was no insertion of this synthetic molecule into themembrane bilayer.

Example 3 Low Temperature Transformation of RBCs by A_(tri)-sp-Ad-DOPE(I) and B_(tri)sp-Ad-DOPE (VI) Synthetic Glycolipids

RBCs are healthier when stored at 4° C., and likewise are believed to behealthier when transformed at 4° C. It was not thought that asignificant rate of insertion of the synthetic glycolipids would occurat 4° C. due to our previous studies (see Comparative Examples) andstudies by others (Schwarzmann, 2000). These studies were performed withnatural glycolipids. Surprisingly these studies did not predict thebehaviour of the synthetic glycolipids of the invention.

Whilst not wishing to be bound by theory, in the studies of Schwarzmannthe low rate of insertion of the natural glycolipids may be due to thephysicochemical properties of the natural glycolipid tail; asphingolipid and a fatty acid.

The diacyl tail of the glycolipid may be important in determining therate of insertion. Certain diacyl tails may retain greater fluidity atlower temperatures. Alternatively, the domain of the plasma membraneinto which the diacyl tail of these glycolipids inserts may retain thisgreater fluidity.

It is known that the sphingolipid tails of natural glycolipidscongregate in rigid domains and these domains may not allow furtherincorporation of glycolipid at low temperatures. Synthetic glycolipidswith cis-desaturated diacyl tails may be favoured for use.

Transformation of RBCS with synthetic glycolipids with different lipidtails was first evaluated (Tables 22 and 24).

TABLE 23 Antisera used to obtain results presented in Tables 24 to 27.Manufacturer Catalogue ref Batch number Expiry date Anti-A Albaclone,SNBTS Z0010770 12 Dec. 2004 BioClone, OCD Experimental 01102 — reagentAnti-B Albaclone, SNBTS Z0110600 27 Apr. 2003 BioClone, OCD Experimental01103 — reagent

TABLE 24 Evaluation of insertion of different lipid tails byagglutination with the relevant antisera. Anti- Transformation solution(μg/mL) Molecule sera 1000 500 250 125 100 60 50 40 30 20 10A_(tri)-sp-Ad-DOPE (I) Alba w+ w+ 0 0 0 Bio 2+ 1+ w+ 0 0 Alba 4+ 3+ 2-3+2+ Bio 4+* 4+* 3+* 3+ DBA 0 B_(tri)-sp-Ad-DOPE (VI) Alba 3+ Bio 3+ Alba2+ 2+ 1+ 0 0 Bio 3+ 2+ 1+ 0 0 A_(tri)-spsp₁-Ad-DOPE (II) Alba 0 0 0 0 0Bio 0 0 0 0 0 Alba 4+ 3+ 2+ 2+ Bio 4+* 3-4+* 3+* 2+ DBA 0A_(tri)-sp-lipid (IV) Alba 0 Bio 0 A_(tri)-sp-Ad-DSPE (III) Alba 0 0 0 00 Bio 0 0 0 0 0 Alba 2-3+ 2-3+ 2+ 2+ Bio 3+ 2-3+ 2+ 2+ DBA 0 *splatter.

Transformation of RBCs with synthetic glycolipids A_(tri)-sp-Ad-DOPE (I)and B_(tri)sp-Ad-DOPE (VI) at 4° C. was then evaluated (Tables 25 to28). These transformations were directed towards the preparation ofcells expressing low levels of A, B or A and B glycotopes (“weak A, Band AB cells”).

For the preparation of weak A and B cells transformation solutions (20μL, A_(tri)sp-Ad-DOPE (I) at 0.08, 0.05 and 0.03 mg/mL, andB_(tri)-sp-Ad-DOPE (VI) at 0.6, 0.3, 0.15, 0.08, 0.05 and 0.03 mg/mL) in1×PBS were mixed with washed, packed group O RBCs (60 μL).

For the preparation of weak AB cells transformation solutions (20 μL,A_(tri)-sp-Ad-DOPE (I) at 0.07, 0.06 and 0.05 mg/mL, andB_(tri)-sp-Ad-DOPE (VI) at 0.3, and 0.2 mg/mL) in 1×PBS were combined inblock titre with washed, packed group O RBCs (60 μL). The combinationswere: A_(tri)sp-Ad-DOPE (I) at 0.07 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.3mg/mL; A_(tri)-sp-Ad-DOPE (I) at 0.07 mg/mL+B_(tri)-sp-Ad-DOPE (VI) at0.2 mg/mL; A_(tri)-sp-Ad-DOPE (I) at 0.06 mg/mL+B_(tri)-sp-Ad-DOPE (VI)at 0.3 mg/mL; A_(tri)-sp-Ad-DOPE (I) at 0.06 mg/mL+B_(tri)-sp-Ad-DOPE(VI) at 0.2 mg/mL; A_(tri)-sp-Ad-DOPE (I) at 0.05mg/mL+B_(tri)-sp-Ad-DOPE (VI) at 0.3 mg/mL; and A_(tri)-sp-Ad-DOPE (I)0.05+B_(tri)-sp-Ad-DOPE (VI) 0.2 mg/mL.

Cells and transformation solutions were placed in a 4° C. fridge.Pipette mixing was performed at intervals. Cells were removed fortesting at intervals against the relevant antisera and were tested inboth washed and unwashed states (i.e. washed samples had thetransformation solution removed).

After 48 hours Celpresol™ was added to the cells so that the finalcells:non-cells ratio was 3:5 (v/v). The cells continued to be tested atintervals. Testing was discontinued after 10 days because cells turnedbrown.

This discolouration could be attributed to a number of factorsincluding: cells were already 21 days old when transformed; 48 hourtransformation was in PBS not Celpresol™ so cells stressed for thistime; and cells may have been mishandled in transit between thetransforming and testing laboratories. This may be mitigated bytransformation of the cells in Celpresol™ as opposed to PBS.

TABLE 25 Diamed results of weak A RBCs transformed at 4° C. againstanti-A. A_(tri)-sp-Ad-DOPE (I) (mg/mL) Washed unwashed Time 0.08 0.050.03 0.08 0.05 0.03  2 hrs 0 0 0 0 0 0  4 hrs 1+ 0 0 2+ 0 0  6 hrs 2+ 00 2+ 0 0  8 hrs 2+ 0 0 2-3+ 0 0 12 hrs 2-3+ 0 0 3+ 1+ 0 24 hrs 3-4+ 1+ 03-4+ 2+ 0 30.5 hr    3-4+ 1+ 0 3-4+ 2+ 0 48 hrs 4+ 2+ 0 4+ 2+ 0 72 hrs4+ 2+ 0 4+ 2-3+ 0 96 hrs 4+ 2-3+ 0 4+ 2-3+ 0 Day 7  3-4+ 2+ 0 Day 103-4+ 2+ 0

TABLE 26 Diamed results of weak B RBCs transformed at 4° C. againstanti-B. B_(tri)-sp-Ad-DOPE (VI) (mg/mL) washed unwashed Time 0.6 0.30.15 0.6 0.3 0.15  2 hrs 0 0 0 0 0 0  4 hrs 0 0 0 1+ 0 0  6 hrs w+ 0 01+ 0 0  8 hrs 2+ 0 0 2+ w+ 0 12 hrs 2+ w+ 0 2-3+ 2+ 0 24 hrs 4+ 3+ 2+ 4+3+ 2+ 30.5 hr    4+ 2-3+ 0 4+ 2-3+ w+ 48 hrs 4+ 3+ 1+ 4+ 3+ 2+ 72 hrs 4+4+ 2+ 4+ 4+ 2+ 96 hrs 4+ 3-4+ 2-3+ 4+ 3-4+ 2-3+ Day 7  4+ 2-3+ 0 Day 104+ 2+ 0

TABLE 27 Diamed results of weak AB RBCs transformed at 4° C. in blocktitre against anti-A. B_(tri)-sp-Ad- A_(tri)-sp-Ad-DOPE (I) (mg/mL) DOPE(VI) washed unwashed Day (mg/mL) 0.07 0.06 0.05 0.07 0.06 0.05 1 0.3 2+1-2+ w+ 2-3+ 2+ 1+ 0.2 2+ 1-2+ 0 2-3+ 2+ 1+ 5 0.3 2+ 1-2+ 1+ 2-3+ 2+1-2+ 0.2 2+ 1-2+ w+ 2-3+ 2+ 1-2+ 8 0.3 2-3+ 2+ 2+ 0.2 2-3+ 2+ 1-2+

TABLE 28 Diamed results of weak AB RBCs transformed at 4° C. in blocktitre against anti-B. B_(tri)-sp-Ad- A_(tri)-sp-Ad-DOPE (I) (mg/mL) DOPE(VI) washed unwashed Day (mg/mL) 0.07 0.06 0.05 0.07 0.06 0.05 1 0.3 3+3+ 2+ 3+ 3+ 2-3+ 0.2 1+ 1-2+ 0 2+ 2+ 1-2+ 5 0.3 2+ 2+ 1+ 2+ 2+ 2+ 0.2 0w+ vw 1+ w+ vw 8 0.3 2+ 2+ 2+ 0.2 1+ 1+ 0

Example 4 Insertion Efficiency of Transformation of RBCs byA_(tri)-sp-Ad-DOPE (I) and B_(tri)-sp-Ad-DOPE (VI) Synthetic Glycolipids

The post-transformation supernatant solutions (from A_(tri)-sp-Ad-DOPE(I) at 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL, and B_(tri)-sp-Ad-DOPE(VI) at 0.6 mg/mL, 20 μL) were added neat and in a 1:2 dilution towashed, packed RBCs (60 μL). The tubes were incubated in a 37° C.waterbath for one hour, with mixing taking place every 15 minutes.

The transformed RBCs were washed 3× with PBS and then suspended inCellstab™ at the appropriate concentration for serology testing.

TABLE 29 Tube serology Pre-trans conc (mg/mL) Score A_(tri)-sp-Ad-DOPE(I) at 0.08 0 1:2 ofA_(tri)-sp-Ad-DOPE (I) at 0.08 0 A_(tri)-sp-Ad-DOPE(I) at 0.05 0 1:2 of A_(tri)-sp-Ad-DOPE (I) at 0.05 0 A_(tri)-sp-Ad-DOPE(I) at 0.03 0 1:2 of A_(tri)-sp-Ad-DOPE (I) at 0.03 0 B_(tri)-sp-Ad-DOPE(VI) at 0.60 vw+ 1:2 of B_(tri)-sp-Ad-DOPE (VI) at 0.60 0

The score given by the post-transformation supernatant solution (fromthe 0.08 mg/mL pre-transformation solution) is not even that of the 0.03mg/mL transformation solution in the first pass (w+). These resultsindicate that >75% of the molecules are inserted into the RBC membraneon the first pass.

In addition, the post-transformation solutions were concentrated 20× andcompared in parallel with the transformation solutions of knownconcentration. Only the post-transformation solutions derived from the0.08 mg/mL A_(tri)-sp-Ad-DOPE (I) and 0.6 mg/mL B_(tri)-sp-Ad-DOPE (VI)solutions were tested.

Post-transformation solutions (20 μL) were dialysed (pore size 500Da)against de-ionised water for 2 days. The samples were left to dry in afumehood for 10 days. At the end of this time they were transferred intoa rotavapor flask and set on the rotavapor to rotate under vacuum withno heat overnight.

Samples were dried in a water bath at 40° C. and washed over intosmaller vessels with chloroform-methanol 2:1 leaving significant amountsof dried cellular material. The chloroform-methanol 2:1 washings weredried down, washed over again into test-tubes with chloroform-methanol2:1 and dried down. These samples were redissolved in 1 mL of 1×PBS andused for transformation experiments. The cellular material in the bottomof the flasks was washed out with water into another set of tubes.

The post-transformation solutions (from A_(tri)-sp-Ad-DOPE (I) at 0.08mg/mL and B_(tri)-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added towashed, packed RBCs (60 μL). In parallel, the transformation solutions(A_(tri)-sp-Ad-DOPE (I) at 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL, andB_(tri)-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added to washed,packed RBCs (60 μL).

The tubes were incubated in a 37° C. waterbath for one hour, with mixingtaking place every 15 minutes. The transformed RBCs were washed 3× withPBS and then suspended in Cellstab™ at the appropriate concentration forserology testing.

TABLE 30 Diamed serology conc (mg/mL) Score A_(tri)-sp-Ad-DOPE (I) at0.08 3+ A_(tri)-sp-Ad-DOPE (I) at 0.05 2+ A_(tri)-sp-Ad-DOPE (I) at 0.031+ From A_(tri)-sp-Ad-DOPE (I) at 0.08 0  B_(tri)-sp-Ad-DOPE (VI) at0.60 4+ From B_(tri)-sp-Ad-DOPE (VI) at 0.60 0 

These results suggest that there are not enough molecules in thepost-transformation solution, even when concentrated 20×, to be detectedby serology.

Example 5 Transformation of Murine RBCs by H_(tri)-sp-Ad-DOPE (VII)Synthetic Glycolipid

Mouse cells were transformed at 37° C. for 1 hour.

TABLE 31 Anti-H reagents used for results in Tables 32 and 33. AntiseraManufacturer Batch Anti-H IgM Japanese Red Cross HIRO-75 UEA LorneLaboratories 11549E D.O.E. 06.2004 Bio-UEA EY Labs 201105-2

TABLE 32 Tube Serology. H Antisera UEA Cells IgM T = 0 T = 20 Bio-UEAMouse RBCs (−control) 0  0  0 Mouse RBCs + 0.01 mg/mL 0 H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs + 0.05 mg/mL 1+ H_(tri)-sp-Ad-DOPE(VII) Mouse RBCs + 0.1 mg/mL 3+ H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs +0.25 mg/mL 4+ 1+ H_(tri)-sp-Ad-DOPE (VII) Mouse RBCs + 1 mg/mL 2+ 2+H_(tri)-sp-Ad-DOPE (VII) Human O RBCs (+control) 4+ 1+ 2/3+ 4+

TABLE 33 Diamed Cells Score Mouse RBCs + 0.01 mg/mL H_(tri)-sp-Ad-DOPE 0(VII) Mouse RBCs + 0.05 mg/mL H_(tri)-sp-Ad-DOPE 0 (VII) Mouse RBCs +0.1 mg/mL H_(tri)-sp-Ad-DOPE  2+ (VII) Mouse RBCs + 0.25 mg/mLH_(tri)-sp-Ad-DOPE  3+ (VII)

Example 6 Transformation of RBCs by Filtered A_(tri)-sp-Ad-DOPE (I)Synthetic Glycolipid

Some A_(tri)-sp-Ad-DOPE (I) had been sterile-filtered through a 0.2 μmfilter. To investigate whether transformation would be the same withthis product a comparative trial was done.

TABLE 34 Anti-A used for results presented in Table 35. ManufacturerCatalogue ref Batch number Expiry date BioClone, OCD Experimentalreagent 01102 —

TABLE 35 Column agglutination of A RBCs transformed with varyingconcentrations of sterile-filtered vs unfiltered A_(tri)-sp-Ad-DOPE (I).Concentration Sterile-filtered Unfiltered (mg/mL) A_(tri)-sp-Ad-DOPE (I)A_(tri)-sp-Ad-DOPE (I) 0.2   4+  4+ 0.1   4+ 3-4+ 0.05 2-3+ 2-3+ 0.01 00 Control 37° C. 0 Control 25° C. 0

These results show no significant difference between the twopreparations of A_(tri)-sp-Ad-DOPE (I) and suggests that filtrationthrough a 0.2 μM filter did not remove molecules or change thecomposition or properties of the fluid to the point that transformationwas affected.

Example 7 Storage of Transformed Cells

To investigate whether storage at 4° C. or 37° C. changed theagglutination results of A_(tri)-sp-Ad-DOPE (I) and natural A glycolipidtransformed O RBCs, identified as “Syn-A” and “Nat-A” cellsrespectively, were divided in two and suspended to 5% in Cellstab™.

One set of cells was stored at 4° C. and the other set of cells wasstored at 37° C. in a waterbath. Agglutination of the stored transformedcells was assessed (Table 36).

TABLE 36 Syn-A A_(tri)-sp-Ad- Nat-A Time Temp DOPE (I) at At 1 At(hours) Platform (° C.) 0.1 mg/mL mg/mL 10 mg/mL Control 0 Tube 3+ 01-2+ 0 20 Column 4 4+ 0 3+ 0 37 4+ 0 3+ 0 44 Column 4 4+ 3+ 0 37 4+ 3+ 0

Example 8 RBC Transformation with A- and B-Antigen Synthetic Glycolipidswith Different Non-Carbohydrate Structures

The water soluble synthetic glycolipids designated A_(tri)-sp-Ad-DOPE(I), A_(tri)-sp₁sp₂-Ad-DOPE (II), A_(tri)-sp-Ad-DSPE (III), andB_(tri)-sp-Ad-DOPE (VI) were prepared according to the method describedin Example 1 with necessary modifications.

Washed packed group O red blood cells (RBCs) (3 parts by volume) and thesynthetic glycolipid solution (1 part by volume, varying concentrations)were added to an eppendorf tube. The tube was incubated in a 37° C.waterbath for one hour, mixing every 15 minutes. The transformed RBCswere washed 3× with PBS and then suspended in Cellstab™ at theappropriate concentration for serology testing.

Tube serology and Diamed gel-card results for RBCs transformed with thedifferent synthetic molecule constructs are provided in Table 38.Results for the stability of the RBCs transformed with the differentsynthetic glycolipids at different concentrations are provided in Tables39 to 44.

TABLE 37 Antisera used for results presented in Tables 38 to 44.Antisera Manufacturer Batch Albaclone anti-A SNBTS Z0010770 - D.O.E12.12.04 Bioclone anti-A Ortho Diagnostics 01102 - D.O.M 16.05.02Albaclone anti-B SNBTS Z0110670 - D.O.E 12.12.04 Bioclone anti-B OrthoDiagnostics 01103 - D.O.M 16.05.02

TABLE 38 Comparison of transformation of RBCs using A-antigen syntheticglycolipids at different concentrations. A Antisera Conc Albacloneanti-A Bioclone anti-A Synthetic mg/mL Tube Diamed Tube DiamedA_(tri)-sp-Ad-DOPE 0.25 n.d.  4+ n.d.  4+ (I) 0.1 n.d. 4+/3+ n.d. 4+/3+0.05 w+  2+  2+  2+ 0.04 w+ n.d.  1+ n.d. 0.03 0 n.d. w+ n.d. 0.02 0n.d. 0 n.d. 0.01 0 0 0 0 A_(tri)-sp-Ad-DSPE 0.25 n.d. 0 n.d. 0 (III) 0.1n.d. 0 n.d. 0 0.05 0 0 0 0 0.04 0 n.d. 0 n.d. 0.03 0 n.d. 0 n.d. 0.02 0n.d. 0 n.d. 0.01 0 0 0 0 A_(tri)-sp₁sp₂-Ad-DOPE 0.25 n.d.  4+ n.d.  4+(II) 0.1 n.d.  4+ n.d. 4+/3+ 0.05 0  3+ 0  3+ 0.04 0 n.d. 0 n.d. 0.03 0n.d. 0 n.d. 0.02 0 n.d. 0 n.d. 0.01 0 0 0 0 Incubated control — 0 n.d. 0n.d. Bench control — 0 n.d. 0 n.d. Abbreviations: n.d. Not determined

TABLE 39 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I)at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL).Agglutination by manual tube serology. Cell Albaclone anti-A Biocloneanti-A storage Concentration of Transformation Solution (mg/mL) Daysolution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 4+ 4+° 4+° 4+°Cellstab ™ 4+ 4+ 3+ 4+° 4+° 4+° 10 Alsevers 3+ 2+ 2+ 4+° 4+° 3+Cellstab ™ 4+° 3+° 2+ 4+° 4+° 4+° 17 Alsevers 4+ 4+ 4+ 4+° 4+° 4+°Cellstab ™ 4+ 4+ 4+ 4+° 4+° 4+° 24 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™4+ 4+ 4+ 4+° 4+ 4+ Abbreviations: ° splatter

TABLE 40 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I)at low concentrations (0.1 mg/mL, 0.05 mg/mL and 0.025 mg/mL).Agglutination by manual tube serology. Cell Albaclone anti-A Biocloneanti-A storage Concentration of Transformation Solution (mg/mL) Daysolution 0.1 0.05 0.025 0.1 0.05 0.025 2 Alsevers 3+/2+ 1+ 1+/w+ 2+2+/1+ 1+ Cellstab ™ 3+/2+ 2+ 1+ 3+/2+ 3+/2+ 2+ 8 Alsevers 2+ 1+ w+ 3+/2+2+ 2+ Cellstab ™ 2+ 1+/w+ vw 3+° 2+ 1+ 15 Alsevers 2+ 1+ 0 3+ 2+ VwCellstab ™ 4+ w+ 0 4+ 4+ 1+ 22 Alsevers 2+ 2+ 0 3+ 2+ w+ Cellstab ™ 4+4+ 1+ 4+ 4+ 1+ 44 Alsevers n.d. n.d. n.d. n.d. n.d. n.d. Cellstab ™ 4+2+ w+ 4+ 2+ w+ Abbreviations: n.d. Not determined ° splatter

TABLE 41 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I)at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL).Agglutination in Diamed gel-cards. Albaclone anti-A Bioclone anti-A CellConcentration of Transformation storage Solution (mg/mL) Day solution 10.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+4+ 4+ 10 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 17Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 24 Alsevers 4+4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 45 Alsevers 4+ 4+ 4+ 4+ 4+4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 59 Alsevers 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+4+ 4+ 4+ 4+ 73 Alsevers Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 88 AlseversCellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ Where there were insufficient cells fortesting, blank spaces have been left.

TABLE 42 Stability trial of RBCs transformed with A_(tri)-sp-Ad-DOPE (I)at low concentrations (0.1 mg/mL, 0.05 mg/mL and 0.025 mg/mL).Agglutination in Diamed gel-cards. Cell Albaclone anti-A Bioclone anti-Astorage Concentration of Transformation Solution (mg/mL) Day solution0.1 0.05 0.025 0.1 0.05 0.025 2 Alsevers 4+ 2+ 0 4+ 3+ 1+ Cellstab ™ 4+2+ 0 4+ 3+ 1+ 8 Alsevers 4+ 3+ 0 4+ 4+ 1+ Cellstab ™ 4+ 3+ 0 4+ 4+ 1+ 15Alsevers 4+ 2+ 0 4+ 3+/2+ 1+ Cellstab ™ 4+ 4+ 0 4+ 4+ 1+ 22 Alsevers 4+3+/2+ 0 4+ 3+ w+ Cellstab ™ 4+ 4+ 0 4+ 4+ 1+ 29 Alsevers 4+ 2+ 0 4+ 3+w+ Cellstab ™ 4+ 3+ 0 4+ 4+ 2+ 43 Alsevers 4+ 3+ w+ 4+ 4+ 2+ Cellstab ™4+ 4+/3+ 0 4+ 4+ 1+ 50 Alsevers 4+ 3+ w+ 4+ 4+ 2+ Cellstab ™ 4+ 3+ 0 4+4+ 1+ 57 Alsevers 4+ 3+/2+ 4+ 4+ Cellstab ™ 4+ 3+ 0 4+ 3+ w+ 63 AlseversCellstab ™ 4+/3+ 2+ 0 4+ 3+ 0 71 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 086 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 0 Where there were insufficientcells for testing, blank spaces have been left.

TABLE 43 Stability trial of RBCs transformed with B_(tri)-sp-Ad-DOPE(VI) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL).Agglutination by manual tube serology. Cell Albaclone anti-B Biocloneanti-B storage Concentration of Transformation Solution (mg/mL) Daysolution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 3+ 3+ 2+ 2+ 1+ 1+ Cellstab ™3+ 2+ 2+ 2+ 2+ 1+ 9 Alsevers 4+ 4+ 2+ 4+ 3+ 2+ Cellstab ™ 4+ 4+ 3+ 4+ 4+2+ 16 Alsevers 4+ 4+ 3+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 23Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 30 Alsevers 3+3+ 2+ 2+ 2+ 2+ Cellstab ™ 4+ 3+ 2+ 3+° 3+° 2+ 37 Alsevers 3+ 2+ 1+ 3+ 2+1+ Cellstab ™ 3+ 3+ 2+/1+ 4+° 3+ 1+ 44 Alsevers 4+ 3+ 1+ 3+ 3+ w+Cellstab ™ 4+ 4+ n.d. 4+ 4+ ^(‡) 51 Alsevers 3+ 3+ 2+ 4+ 3+ 2+Cellstab ™ 4+ 4+ n.d. 4+ 4+ 2+ Abbreviations: ° splatter

TABLE 44 Stability trial of RBCs transformed with B_(tri)-sp-Ad-DOPE(VI) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL).Agglutination in Diamed gel-cards. Cell Albaclone anti-B Bioclone anti-Bstorage Concentration of Transformation Solution (mg/mL) Day solution 10.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 2+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+4+ 2+ 9 Alsevers 4+ 4+ 2+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 16Alsevers 4+ 4+ 2+ 4+ 4+ 1+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 23 Alsevers 4+4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 30 Alsevers 4+ 4+ 3+ 4+ 4+3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 37 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™4+ 4+ 3+ 4+ 4+ 3+ 44 Alsevers 4+ 4+ 2+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+4+ 4+/3+ 51 Alsevers 4+ 4+ 2+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 58Alsevers 4+ 1+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 72 Alsevers 4+ 2+ 4+3+ Cellstab ™ 4+ 4+ 3+/2+ 4+ 4+ 3+ 87 Alsevers Cellstab ™ 4+ 4+/3+ 1+ 4+4+/3+ 2+/1+ 116 Alsevers Cellstab ™ 4+ 3+ 0 4+ 4+/3+ 1+ Where there wereinsufficient cells for testing, blank spaces have been left.

Example 9 Red Blood Cell Transformation with H-Antigen SyntheticGlycolipids

The water soluble synthetic glycolipids designated H_(tri)-sp-Ad-DOPE(VII), H_(di)-sp-Ad-DOPE (VIII) and Galβ-sp-Ad-DOPE (IX) were preparedaccording to the method described in Example 1 with necessarymodifications.

Washed packed mouse RBCs (3 parts by volume) and the syntheticglycolipid solutions (1 part by volume of varying concentrations) wereadded to an eppendorf tube. The tube was incubated in a 37° C. waterbathfor one hour, mixing every 15 minutes. The transformed RBCs were washed3× with PBS and then suspended in Cellstab™ at the appropriateconcentration for serology testing.

Tube serology and Diamed gel-card results for RBCs transformed with thedifferent synthetic glycolipids are presented in Table 46. The resultsshow that three sugars (H_(tri)) are required for detection by anti-HIgM, at least by the reagent used.

TABLE 45 Antisera used for results presented in Table 46. AntiseraManufacturer Batch Anti-H IgM Japanese Red Cross HIRO-75 UEA LomeLaboratories 11549E D.O.E. 06.2004 Bio-UEA EY Labs 201105-2

TABLE 46 Comparison of transformation of RBCs using H-antigen syntheticglycolipids with different glycotopes made to different concentrations.H Antisera UEA Bio- Conc IgM Tube Tube UEA Synthetic mg/mL Tube DiamedT0 T20 Tube H_(tri)-sp-Ad-DOPE (VII) 1 n.d. n.d. 2+ n.d. 2+ 0.25 4+ 3+n.d. n.d. 1+ 0.1 3+ 2+ n.d. n.d. n.d. 0.05 1+ 0 n.d. n.d. n.d. 0.01 0 0n.d. n.d. n.d. H_(di)-sp-Ad-DOPE (VIII) 0.25 0 n.d. n.d. n.d. n.d. 0.1 0n.d. n.d. n.d. n.d. 0.05 0 n.d. n.d. n.d. n.d. 0.01 0 n.d. n.d. n.d.n.d. Galβ-sp-Ad-DOPE (IX) 0.25 0 n.d. n.d. n.d. n.d. 0.1 0 n.d. n.d.n.d. n.d. 0.05 0 n.d. n.d. n.d. n.d. 0.01 0 n.d. n.d. n.d. n.d. Human Ocells — 4+ n.d. 1+ 2/3+ 4+ Incubated control — 0 n.d. 0 0 n.d. Benchcontrol — 0 n.d. n.d. n.d. n.d. Abbreviations: n.d. Not determined

Example 10 Insertion of H_(di)-sp-Ad-DOPE (VIII) and Galβ-sp-Ad-DOPE(IX) Synthetic Glycolipids into Murine Red Blood Cells

The water soluble synthetic glycolipids designated H_(di)-sp-Ad-DOPE(VIII) and Galβ-sp-Ad-DOPE (IX) were prepared according to the methoddescribed in Example 1 with necessary modifications.

Murine RBCs were washed 3× in 1×PBS. 30 μl of packed RBCs were combinedwith 30 μl of H_(di)-sp-Ad-DOPE (VIII), and 30 μl of packed RBCs werecombined with 30 μl Galβ-sp-Ad-DOPE (IX), respectively. Both syntheticmolecule constructs were at a concentration of 1.0 mg/ml. 30 μl of 1×PBSwas added to 30 μl of packed RBCs to act as the control group. Cellswere incubated for 90 minutes in a 37° C. shaking water-bath. RBCs werewashed 3× in 1×PBS.

Three groups of packed RBCs were incubated with an equal volume oflectin UEA-1 for 30 minutes at room temperature. The lectin was preparedin 1×PBS at a concentration of 0.1 mg/ml. 50 μl of a 3% cell suspensionwas spun for 15 seconds in an Immunofuge at low speed. Results were readby tube serology. The results are presented in Table 48. The resultsshow that neither anti-H IgM nor UEA-1 detects two sugars (H_(di)).

TABLE 47 Antisera used for results presented in Table 48. AntiseraManufacturer Batch Biotest anti-H Biotest AG UEA EY Labs 201105-2

TABLE 48 Murine RBCs transformed with Galβ-sp-Ad-DOPE orH_(di)-sp-Ad-DOPE, assessed by agglutination. Cell Type InsertedMolecule UEA-1 Mouse IgM^(H) Murine RBC Galβ (1 mg/ml) 0 n.d. Murine RBCH_(di) (1 mg/ml) 0 0 Murine RBC Control (PBS) 0 0 Human RBC Control(PBS) 4+  3+ Abbreviations: n.d. Not determined

Example 11 Preparation of Sensitivity Controls

The synthetic glycolipids of the invention may be used in thepreparation of “sensitivity controls” (also referred to as “qualitycontrol cells”, “serology controls”, or “process controls”) as describedin the specification accompanying international application no.PCT/NZ02/00214 (WO 03/034074). The synthetic glycolipids provide theadvantage that the transformation of the RBCs may be achieved at reducedtemperatures.

RBC Transformation Solutions

Two stock solutions are used:

-   -   Solution 1: 1 mg/mL A_(tri)sp-Ad-DOPE (I) suspended in        Celpresol™ solution.    -   Solution 2: 5 mg/mL B_(tri)sp-Ad-DOPE (VI) suspended in        Celpresol™ solution.

Glycolipids are manufactured in a white dry powder. Glycolipids in thisform (enclosed in a sealed container under a controlled temperature) arestable for an indefinite period of time. The glycolipids are suspendedin solution (e.g. Celpresol™) by weight in order to formulate thetransformation solutions.

Once the transformation solutions are received at CSL, they are filtered(through a MILLEX®-GV 0.22μ filter unit) under aseptic conditions.

Processing of RBCs

RBC donations are processed using a continuous flow centrifuge washerunder aseptic conditions. RBC donations are washed in buffered salinefollowed by Celpresol™ solution. The PCV of the RBC donations ismeasured on a Beckman Coulter AcT Diff analyser. The donations are thenadjusted to a packed cell volume (PCV) of 50% with the addition ofCelpresol™.

Transformation of RBCs to Provide “Weak AB Cells”

RBCs are washed in buffered saline and Celpresol™. The cells aresuspended in Celpresol™ solution to a PCV of >50%. The PCV of red cellsis measured using a Beckman Coulter AcT Diff. The mass of the red cellsolution is weighed.

The amount of A_(tri)-sp-Ad-DOPE (I), B_(tri)-sp-Ad-DOPE (VI) andCelpresol™ for transformation is calculated using the followingequations:

$a = \frac{P \times F}{S}$ $b = \frac{P \times F}{S}$c = P − (1 − P) − a − b

where

-   -   a=amount of A_(tri)-sp-Ad-DOPE (I) to be added per 1 mL of red        cells (mL)    -   b=amount of B_(tri)-sp-Ad-DOPE (VI) to be added per 1 mL of red        cells (mL)    -   c=amount of Celpresol™ to be added per 1 mL of red cells (mL) to        dilute cells to 50% PCV    -   P=PCV of red cell solution    -   F=Final desired concentration of glycolipid    -   S=Concentration of stock glycolipid solution

To determine the amount of glycolipid and Celpresol™ to add to a bulksample of red cells, multiply each of a, b and c by the red cell volume.Add A_(tri)-sp-Ad-DOPE (I), B_(tri)-sp-Ad-DOPE (VI) and Celpresol™ tothe red cell bulk sample aseptically.

Incubate the sample for 3 hours at 20° C. under controlled temperatureconditions and constant gentle agitation. At the end of the 3 hourperiod, aseptically remove a sample of red cells and test the sample toconfirm transformation of the RBCs. Perform blood grouping using tube,tile and column agglutination technology (CAT) techniques.

Incubate the red cell sample for 3 hours at 2-8° C. under controlledtemperature conditions and constant gentle agitation for 18 hours. Atthe end of the 3 hour period, aseptically remove a sample of red cellsand test the sample to confirm transformation of the red cells. Performblood grouping using tube, tile and CAT techniques.

Wash the transformed red cells using a continuous flow centrifugemethod, under aseptic conditions using Celpresol™ solution. Measure thePCV of the washed red cells and adjust to 50% PCV by the addition ofCelpresol™ solution.

Formulation and Dispensing

Aseptically combine a volume of the transformed RBCs with a volume ofsimulated plasma diluent (SPD). The plasma may contain monoclonal andpolyclonal antibodies. Antibodies are selected according to the desiredcharacteristics of the sensitivity controls. The plasma may additionallycontain tartrazine and bovine serum albumin.

Blood grouping and antibody screening is performed on the bulk samplesusing tube, tile and CAT techniques. The transformed RBC-SPD blend isthen aseptically dispensed into BD Vacutainer tubes and the tubeslabelled accordingly.

Validation Testing

Weak AB cells produced by the use of synthetic glycolipids (designatedA_(w)B_(w) in Tables 51 to 53) were used to validate a range of testingplatforms in parallel with naturally occurring weak

A, weak B and weak AB cells.

TABLE 49 Reagents and cards used in validation testing. Method ReagentTube Epiclone Tile Epiclone Ref Manufacturer and type Batch Expiry CAT 1OCD BioVue ABD/Rev ABR528A 16 Jun. 2005 CAT 2 OCD BioVue ABD/Rev ABR521A06 May 2006 CAT 3 OCD BioVue ABD/ABD ACC255A 24 May 2005 CAT 4 DiamedID-MTS 50092.10.02 April 2005 CAT 5 Diamed ID-MTS Donor typing51051.05.04 March 2005 CAT 6 Diamed ID-MTS Recipient typing 50053.07.02April 2005 CAT 7 Diamed ID-MTS Cord typing 50961.08.03 July 2005

TABLE 50 Testing platform methodology for validation testing. Tile 1drop 3% cells, 2 drops reagent, 15 min @ RT in moist chamber. Tube 2drops @ RT, 10 min. ID-MTS As per manufacturers instructions usingDil-2. BioVue As per manufacturers instructions using 0.8% RCD.

TABLE 51 Validation results across all methods against anti-A. Testingplatform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1A_(x) w+ 0 2+ 1+ 0 0 0 0 2 A_(x) w+ 0 2+ 2+ 0 0 0 0 3 A₁B 4+ 4+ 4+ 4+ 4+4+ 4+ 4+ 4+ 4 A_(x) w+ 0 2+ 2+ 0 0 0 0 5 A₂B 3+ 3+ 4+ 3+ 3+ 1+ 2+ 3+ 6A_(x) w+ 0 2+ 2+ 0 0 0 0 7 A_(x) 1+ 0 2+ 2+ 0 0 0 0 8 A_(x) w+ 0 2+ 2+ 00 0 0 9 A_(x) 0 0 1+ 1+ 0 0 0 0 10 A_(x) w+ 0 2+ 2+ 0 0 0 0 11 A₃ 4+ 4+4+ 3+ 3+ 1+ 1+ 3+ 12 A₃B 3+ 3+ 3+ 3+ 2+ w+ w+ 2+ 13 B₃ 0 0 0 0 0 0 0 0 014 B₃ 0 0 0 0 0 0 0 0 0 15 A_(w)B_(w) 2+ 2+ 2+ 2+ 2+ 0 0 0 0

TABLE 52 Validation results across all methods against anti-B. Testingplatform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1A_(x) 0 0 0 0 0 0 0 0 2 A_(x) 0 0 0 0 0 0 0 0 3 A₁B 4+ 4+ 4+ 4+ 4+ 4+ 3+3+ 4+ 4 A_(x) 0 0 0 0 0 0 0 0 5 A₂B 4+ 4+ 4+ 4+ 4+ 3+ 3+ 4+ 6 A_(x) 0 00 0 0 0 0 0 7 A_(x) 0 0 0 0 0 0 0 0 8 A_(x) 0 0 0 0 0 0 0 0 9 A_(x) 0 00 0 0 0 0 0 10 A_(x) 0 0 0 0 0 0 0 0 11 A₃ 0 0 0 0 0 0 0 0 12 A₃B 4+ 4+4+ 4+ 4+ 4+ 4+ 4+ 13 B₃ 2+ 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+ 14 B₃ 2+ 2+ 2+ 2+ 2+2+ 1+ 1+ 2+ 15 A_(w)B_(w) 3+ 3+ 1+ 1+ 1+ 0 0 0 0

TABLE 53 Validation results across all methods against anti-AB. Testingplatform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1A_(x) 3+ 2+ 2+ 2 A_(x) 4+ 2+ 3+ 3 A₁B 4+ 4+ 4+ 4 A_(x) 3+ 2+ 3+ 5 A₂B 4+4+ 4+ 6 A_(x) 4+ 4+ 3+ 7 A_(x) 4+ 4+ 3+ 8 A_(x) 3+ 4+ 3+ 9 A_(x) 4+ 2+2+ 10 A_(x) 3+ 4+ 3+ 11 A₃ 4+ 4+ 4+ 12 A₃B 4+ 4+ 4+ 13 B₃ 2+ 2+ 2+ 14 B₃2+ 2+ 2+ 15 A_(w)B_(w) 3+ 3+ 3+

Example 12 Attachment of Modified Embryos to Transformed EndometrialCells

The ability to effect qualitative and quantitative differences in thecell surface antigens expressed by cell types other than RBCs wasinvestigated. The ability to enhance the adhesion of embryos toendometrial cells was adopted as a model system.

The synthetic molecules may be used as synthetic membrane anchors and/orsynthetic molecule constructs. Therefore, they may also be employed inthe method of enhancing embryo implantation as described ininternational patent application no PCT/NZ2003/000059 (published as WO03/087346) which is incorporated by reference.

Endometrial Cell Transformation

Insertion of Water Soluble Synthetic Molecule Construct

A single cell suspension of endometrial epithelial cells was prepared.The endometrial cells were washed 3× by resuspending in CMF HBSS andcentrifuging at 2000 rpm for 3 minutes.

The washed cell preparation was resuspended in 50 μl of M2.

Micro-centrifuge tubes each containing a 50 μl solution of 5 M/mlendometrial cells were prepared. To separate tubes of endometrial cells50 μl of synthetic glycolipids A_(tri)-sp-Ad-DOPE (I) orB_(tri)-sp-Ad-DOPE A (VI), or 50 μl M2 were added to the control cells.The cells were incubated for 90 minutes at 37° C. on a mixer. Theendometrial cells were washed 3× by resuspending in CMF HBSS media andcentrifuging at 2000 rpm for 3 minutes. The washed cell preparation wasresuspended in 50 μl of M2.

Test For Insertion Using Fluorescent Probe:

50 μl of corresponding primary murine monoclonal antibody was added toeach tube. Each tube was incubated at room temperature for 10 minutes.Cells were washed 3× in M2 media. 10 μl of mouse anti-IgG FITC was addedto each tube. Tubes were incubated at room temperature in darkconditions for 10 minutes. Endometrial cells were mounted on glassslides and viewed under a fluorescence microscope.

Test for Direct Agglutination:

5 μl of each group of cells was placed onto separate microscope slides.To each 5 μl drop of cells 5 μl of a corresponding antibody was added.The cells were gently mixed on the slide for 2 minutes. Agglutinationwas visualised under the microscope. The results are presented in Table55.

TABLE 54 Antisera used for results presented in Table 55. AntiseraManufacturer Bioclone anti-A Ortho Diagnostics 01102 D.O.M. 16.05.02Bioclone anti-B Ortho Diagnostics Developmental reagent

TABLE 55 Endometrial cells transformed with A_(tri)-sp-Ad-DOPE (I) orB_(tri)-sp-Ad-DOPE A (VI), as visualised using fluorescence.Agglutination Fluorescence score reaction by after incubation withmicroscopic Cell Type Inserted Antigen 1° antibody IgFITC Probevisualisation Endometrial A_(tri)-sp-Ad-DOPE Anti-A Bioclone 4+ 4+ cells(I) (1 mg/ml) Endometrial B_(tri)-sp-Ad-DOPE Anti-B Bioclone 1+ 3+ cells(VI) (1 mg/ml) Endometrial Control (M2 Anti-A Bioclone 0  0  cellsmedia)

Embryo Modification

Insertion of Water Soluble Synthetic Molecule Construct:

The embryo zona pellucida was removed by treating embryos with 0.5%pronase in a 37° C. oven for 6 minutes or until all zonas were removed.Micro-drops were prepared by adding 5 μl of synthetic glycolipidA_(tri)-sp-Ad-DOPE (I) or B_(tri)-sp-Ad-DOPE (VI), at a concentration of1 mg/mL to a 45 μl drop of M2 media overlaid with mineral oil. Allembryo groups were incubated in the 50 μl micro-drops for 1 hour at 37°C. Embryos from experimental and control groups were washed 3× with M2media.

Test for Insertion:

Embryos from experimental and control groups were placed into amicro-drop of corresponding antibody and incubated for 30 min at 37° C.Embryos from experimental and control groups were washed 3× with M2media.

Embryos from all experimental and control groups were placed intomicro-drops of anti-mouse Ig FITC (1:50 dilution anti-mouse Ig FITC inM2) and incubated for 30 min at 37° C. Embryos from experimental andcontrol groups were washed 3× with M2 media. Embryos were mounted onmicroscope slides in a 5 μl drop of M2 and the drops overlaid with oil.

The slides were viewed under a fluorescence microscope. Results arepresented in Tables 56 and 57. The negative result for transformationwith B_(tri)-sp-Ad-DOPE (VI) is attributed to a lack of 1° antibodysensitivity.

TABLE 56 Embryos transformed with A_(tri)-sp-Ad-DOPE (I) as visualisedusing fluorescence. Fluorescence score Embryo after incubation withMorphology 24 hr Cell Type Inserted Antigen 1° antibody IgFITC Probepost insertion Embryos A_(tri)-sp-Ad-DOPE (I) Anti-A Bioclone 2+/1+Appeared viable Embryos Control Anti-A Bioclone 0 Appeared viable

TABLE 57 Embryos transformed with A_(tri)-sp-Ad-DOPE (I) orB_(tri)-sp-Ad-DOPE (VI), as visualised using fluorescence. Fluorescencescore Embryo after incubation with Morphology 24hr Cell Type InsertedAntigen 1° antibody IgFITC Probe post insertion EmbryosA_(tri)-sp-Ad-DOPE Anti-A Bioclone  2+ n.d. (I) EmbryosB_(tri)-sp-Ad-DOPE Anti-B Bioclone 0 n.d. (VI) Embryos Control (M2Anti-A Bioclone 0 n.d. media) Abbreviations: n.d. Not determined

Enhanced Attachment Transformed Endometrial Cells to Modified Embryos

Modified embryos (BioG-Avidin-BioIgG^(B) and BioG-Avidin-BioIgM^(A))were prepared in accordance with the methods described in thespecification accompanying the international application no.PCT/NZ03/00059 (published as WO03/087346).

Two concave glass slides were prepared, one with two wells of syntheticglycolipid A_(tri)-sp-Ad-DOPE (I) inserted endometrial cells and theother with two wells of synthetic glycolipid B_(tri)-sp-Ad-DOPE (VI)inserted endometrial cells.

The two groups of embryos were transferred to each of the concave glassslides:

-   -   Slide 1 A_(tri)/IgG^(B) embryos        -   A_(tri)/IgM^(A) embryos    -   Slide 2 B_(tri)/IgG^(B) embryos        -   B_(tri)/IgM^(A) embryos

The embryos were surrounded with endometrial cells. The wells werecovered with mineral oil and incubated for 15 minutes at 37° C. Using awide bore handling pipette each group of embryos were carefullytransferred to a fresh drop of M2 media. The embryos were gently washed.The embryos were gently transferred into 2 μL of M2 media on a markedmicroscope slide. Each drop was overlaid with mineral oil

Under a central plane of focus on an Olympus microscope the number ofendometrial cells adhered to the embryos in each group was assessed. Thenumber of cells adhered to each embryo was recorded. Results arepresented in Table 58.

TABLE 58 Endometrial cells transformed with A_(tri)-sp-Ad-DOPE (I) orB_(tri)-sp-Ad-DOPE (VI), and embryos modified withBioG-Avidin-BioIgG^(B) or BioG-Avidin-BioIgM^(A). Assessment byattachment of endometrial cells to embryos. Transformed Average numberof endometrial endometrial cells attached Cell Type cells Modifiedembryos to modified embryos Endometrial A_(tri)-sp-Ad-DOPEBioG-Avidin-BioIgG^(B) 2.3 cells (I) BioG-Avidin-BioIgM^(A) 7.25Endometrial B_(tri)-sp-Ad-DOPE BioG-Avidin-BioIgG^(B) 6.7 cells (VI)BioG-Avidin-BioIgM^(A) 3.4

Where in the foregoing description reference has been made to integersor components having known equivalents then such equivalents are hereinincorporated as if individually set forth.

Although the invention has been described by way of example and withreference to possible embodiments thereof it is to be appreciated thatimprovements and/or modification may be made thereto without departingfrom the scope or spirit of the invention.

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1-187. (canceled)
 188. A synthetic molecule construct of the structureF-3₁-S₂-L where: F is a mono-, di-, tri- or oligo-saccharide; S₁ is2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl; S₂ is—CO(CH₂)₂CO—, —CO(CH₂)₃CO—, —CO(CH₂)₄CO— or —CO(CH₂)₅CO—; and L is adiacyl- or dialkyl-glycerophospholipid.
 189. The synthetic moleculeconstruct according to claim 188 where the construct includes thesubstructure:

where: n=3 to 5 and M is H.
 190. The synthetic molecule constructaccording to claim 188 where F is selected from the group consisting of:GalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ;NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ;Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ;Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ;Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc;GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; orGalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc.
 191. The synthetic moleculeconstruct according to claim 188 where F is selected from the groupconsisting of: GalNAcα1-3(Fucα1-2)Galβ and Galα1-3(Fucα1-2)Galβ. 192.The synthetic molecule construct according to claim 188 where S₁ is3-aminopropyl.
 193. The synthetic molecule construct according to claim188 where L is selected from the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE).
 194. Asynthetic molecule construct:

designated A_(tri)-sp-Ad-DOPE (I).
 195. A synthetic molecule construct:

designated A_(tri)-sp-Ad-DS PE (III).
 196. A synthetic moleculeconstruct:

designated B_(tri)-sp-Ad-DOPE (VI).
 197. A synthetic molecule construct:

designated H_(tri)-sp-Ad-DOPE (VII).
 198. A synthetic moleculeconstruct:

designated H_(di)-sp-Ad-DOPE (VIII).
 199. A synthetic moleculeconstruct:

designated Galβ-sp-Ad-DOPE (IX).
 200. A synthetic molecule construct:

designated Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc-sp-Ad-DOPE (XII). 201.A synthetic molecule construct:

designated Fucα1-2Galβ1-3(Fucα1-4)GlcNAc-sp-Ad-DOPE (XIII).